Pathogen resistance in plants

ABSTRACT

The present disclosure provides an isolated, recombinant, or synthetic polynucleotide comprising a FIT1 protein, and homologs, fragments, and variations thereof. The disclosure further relates to transgenic plants, plant parts, and plant cells comprising one or more of these polynucleotides, and exhibit resistance or tolerance to a pathogen, such as  Phakopsora pachyrhizi . The disclosure further relates to methods of genetically engineering a pathogen resistance or tolerance trait in a plant, plant part, or plant cell, comprising targeted gene editing of a FIT1 homolog, and plants produced therefrom. The disclosure further relates to methods for identifying new functional FIT1 genes and/or alleles thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/512,329 filed on Oct. 27, 2021 (U.S. Pat. No. 11,530,419; issue dateDec. 20, 2022), which claims priority to and benefit of U.S. ProvisionalApplication No. 63/108,023, filed on Oct. 30, 2020, which are herebyincorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under 1844088 by theNational Science Foundation. The Government has certain rights to thisinvention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:FOTI_001_02US_SeqList_ST26.xml, date recorded: Dec. 1, 2022, file size150,889 bytes).

TECHNICAL FIELD

The disclosure relates to the identification and use of nucleic acidsequences for pathogen resistance in plants.

BACKGROUND

Pathogen effector proteins often convergently evolve to target the sameor similar plant host proteins to promote virulence. Such proteins maybe present in a diverse range of plant pathogens including but notlimited to fungi, oomycetes, bacteria, nematodes or viruses.

An example of one such pathogen that causes harm to plants by secretingan effector protein is the obligate biotrophic fungus Phakopsorapachyrhizi (and to a lesser extent, the closely related fungusPhakopsora meibomiae), which causes Asian soybean rust (ASR). Whilesoybeans make up the primary commercial crop affected by ASR, Phakopsorainfects leaf tissue from a broad range of leguminous plants, includingat least 17 genera (Slaminko et al., 2008). In general, rust fungi(order Pucciniales) constitute one of the most economically importantgroups of plant pathogens because of their larger range of host andgenetic diversity. There are more than 6000 species of rust fungi thatcause harm to many plant species, such as wheat (Puccinia spp.), commonbean (Uromyces appendiculatus), soybean (Phakopsora pachyrhizi), andcoffee (Hemileia vastatrix) (Aime et al., 2006).

Infection in commercial crops requires application of variousfungicides, which are costly and not always effective. In Brazil alone,control of ASR costs $2 billion annually. Thus, there is a need forplant varieties that are resistant to fungal pathogens.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY

The present disclosure provides for an isolated, recombinant, orsynthetic polynucleotide comprising a nucleic acid sequence encoding afunctional FIT1 protein homologous to SEQ ID NO: 2. In some embodiments,the polynucleotide encodes a protein having at least 70% identity to SEQID NO: 2. In some aspects, the protein is selected from the groupconsisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 andfunctional homologs thereof. In some aspects, the isolated, recombinant,or synthetic polynucleotide comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19 complements thereof, fragments thereof, and sequences at least 70%identical thereto. The disclosure further relates to genetic constructscomprising one or more of these sequences, and transgenic plants, plantparts, or plant cells comprising one or more of these sequences, whereinthe plant, plant part, or plant cell is resistant or tolerant to apathogen.

In another embodiment, the disclosure teaches a method of producing aplant, plant part, or plant cell having resistance or tolerance to apathogen, wherein the method comprises transforming a plant, plant part,or plant cell with a polynucleotide encoding a functional FIT1 protein.In some aspects, the nucleotide sequence encoding the FIT1 protein hasbeen codon optimized. In some aspects, the FIT1 protein comprises aselected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20 and functional homologs thereof, or is encoded by anisolated, recombinant, or synthetic polynucleotide selected from thegroup consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,complements thereof, fragments thereof, and sequences at least 70%identical thereto.

In another embodiment, the disclosure teaches a method of geneticallyengineering a pathogen resistance or tolerance trait in a plant, plantpart, or plant cell, comprising providing a plant species that issusceptible to a pathogen, identifying within the genome of the plantspecies a homolog of FIT1, wherein said homolog does not mediate AvrFIT1recognition; and genetically modifying a plant, plant part, or plantcell from the susceptible plant species with targeted gene editing,wherein said targeted gene editing is directed at the FIT1 homolog, andwherein said targeted gene editing enables the FIT1 homolog to recognizeAvrFIT1 and confers resistance or tolerance to a pathogen.

The disclosure further relates to genetically modified plants, plantparts, or plant cells produced by the methods disclosed herein, whereinthe plant, plant part, or plant cell exhibits resistance or tolerance toa pathogen. In some aspects, the pathogen is a fungus from the orderCantharellales or Pucciniales. In some aspects, the fungal pathogen isRhizoctonia solani, Melampsora spp., Phakopsora pachyrhizi, Phakopsorameibomiae, Phakopsora euvitis, Phakopsora spp., Puccinia spp., Uromycesspp., Austropuccinia spp., Cronartium spp. or Hemileia vastatrix. Insome aspects, the plant, plant part, or plant cell is in the subfamilyPapilionoideae. In some aspects, the plant, plant part, or plant cell isAlysicarpus spp., Astragalus spp., Baptisia spp., Cajanus spp.,Calopogonium spp., Caragana spp., Centrosema spp., Cologania spp.,Crotalaria spp., Desmodium spp., Genista spp., Glycine spp., Glycyrrhizaspp., Indigofera spp., Kummerowia spp., Lablab spp., Lathyrus spp.,Lespedeza spp., Lotus spp., Lupinus spp., Macroptilium spp., Macrotylomaspp., Medicago spp., Neonotonia spp., Pachyrhizus spp., Pisum spp.,Phaseolus spp., Pseudovigna spp., Psoralea spp., Robinia spp., Sennaspp., Sesbania spp., Strophostyles spp., Tephrosia spp., Teramnus spp.,Trifolium spp., Vicia spp., Vigna spp., or Voandzeia spp.

The disclosure further relates to methods for identifying a functionalFIT1 gene and/or allele thereof comprising isolating a FIT1 homolog orallele thereof; expressing said FIT1 homolog or allele thereof incombination with an effector protein produced by Phakopsora pachyrhiziin a plant, plant part, or plant cell; and assaying said plant, plantpart, or plant cell for an immune response. In some aspects, theeffector protein comprises SEQ ID NO: 24, SEQ ID: 26, or sequences atleast 90% identical thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The accompanying figures, which are incorporated herein and form a partof the specification, illustrate some, but not the only or exclusive,example embodiments and/or features. It is intended that the embodimentsand figures disclosed herein are to be considered illustrative ratherthan limiting.

FIG. 1 shows a phylogenetic tree of AvrFIT1 homologs identified byperforming a BLAST® search of the NCBI protein database. AdditionalAvrFIT1 sequences were obtained from published Phakopsora pachyrhizitranscriptomes and proteomes. The obtained sequences were aligned usingClustal Omega and manually filtered to remove redundant and incompletesequences. A maximum likelihood phylogenetic tree was constructed fromthe aligned protein sequences.

FIG. 2 shows photographs of plants inoculated with Phakopsora spores attwo to four weeks of age. The images were taken at 14 to 21 days postinoculation. Presence of FIT1 in the plant genome correlates withresistance to ASR.

FIG. 3A shows a phylogenetic tree of homologs of Vigna unguiculataVu01g041300.1 (VuFIT1) (SEQ ID NO: 2) identified by performing a BLAST®search and conducting a protein alignment. This figure shows putativeFIT1 orthologs in Vigna radiata XP_014501487.1 (SEQ ID NO: 4), Vignaangularis XP_017423114.1 (SEQ ID NO: 12), Phaseolus acutifolius005G036500.1 (SEQ ID NO: 10), Phaseolus lunatus Pl05g0000042900.1 (SEQID NO: 6), Phaseolus vulgaris PvUI111.05g037200.1 (SEQ ID NO: 8), Lablabpurpureus c22535_g1_ i3 (SEQ ID NO: 14), Mucuna pruriens RDX66260.1 (SEQID NO: 16), Cajanus cajan XP_020211972.1 (SEQ ID NO: 18), and Abrusprecatorius XP_027357710.1 (SEQ ID NO: 20). Also shown is a paralog ofFIT1 from Vigna unguiculata Vu01g041400.1 (SEQ ID NO: 22) believed to bethe result of a recent duplication which accumulated mutations and isthus non-functional. The phylogenetic tree was rooted using paralogs ofFIT1 that do not function in AvrFIT1 perception. Activity of each FIT1homolog to function for recognition of AvrFIT1a and AvrFIT1b, based ontransient assays, is indicated. nt=not tested.

FIG. 3B shows a protein alignment of the amino acid sequences listed inFIG. 3A, specifically of the Vigna unguiculata allele of FIT1 (VuFIT1)(SEQ ID NO: 2), the Vigna unguiculata close paralog of FIT1 (VuFIT1b)(SEQ ID NO: 22), Vigna angularis allele of FIT1 (VaFIT1) (SEQ ID NO:12), the Vigna radiata allele of FIT1 (VrFIT1) (SEQ ID NO: 4), thePhaseolus acutifolius allele of FIT1 (PaFIT1) (SEQ ID NO: 10), thePhaseolus vulgaris allele of FIT1 (PvFIT1) (SEQ ID NO: 8), the Phaseoluslunatus allele of FIT1 (PlFIT1) (SEQ ID NO: 6), the Lablab purpureusallele of FIT1 (LpFIT1) (SEQ ID NO: 14), the Cajanus cajun allele ofFIT1 (CcFIT1) 1 (SEQ ID NO: 18), the Mucuna pruriens allele of FIT1(MpFIT1) (SEQ ID NO: 16), and the Abrus precatorius allele of FIT1(ApFIT1) (SEQ ID NO: 20).

FIG. 3C shows a phylogenetic tree of homologs of Vigna unguiculata FIT1in Glycine identified by performing a BLAST® search and constructing aprotein alignment and phylogenetic tree of the resulting sequences. Thetree was rooted using an outgroup of distantly related TIR-NLR proteinsfrom non-legumes.

FIG. 4 shows a map of an example DNA construct comprising VuFIT1 (SEQ IDNO: 1) that can be used for transformation of a plant cell, selection oftransformed cells, and expression of FIT1.

FIGS. 5A-5D depict results showing transient expression of various FIT1alleles in leaf tissue from a plant lacking an endogenous FIT1. FIG. 5Ashows the Vigna unguiculata allele of FIT1 (VuFIT1), the Phaseoluslunatus allele of FIT1 (PlFIT1), the Vigna radiata allele of FIT1(VrFIT1), the Vigna unguiculata close paralog of FIT1 (VuFIT1b), andAvrFIT1a. FIG. 5B shows Vigna angularis allele of FIT1 (VaFIT1), thePhaseolus vulgaris allele of FIT1 (PvFIT1), the Lablab purpureus alleleof FIT1 (LpFIT1), the Abrus precatorius allele of FIT1 (ApFIT1), andAvrFIT1a. FIG. 5C shows Vigna unguiculata allele of FIT1 (VuFIT1), thePhaseolus lunatus allele of FIT1 (PlFIT1), the Vigna radiata allele ofFIT1 (VrFIT1), the Vigna unguiculata allele of FIT1 (VuFIT1b), andAvrFIT1b. FIG. 5D shows Vigna angularis allele of FIT1 (VaFIT1), thePhaseolus vulgaris allele of FIT1 (PvFIT1), the Lablab purpureus alleleof FIT1 (LpFIT1), the Abrus precatorius allele of FIT1 (ApFIT1), andAvrFIT1b.

FIGS. 6A-6H depict wild type soybean leaves (lacking FIT1) (FIG. 6A-6D)and leaves from soybean plants expressing FIT1 (FIG. 6E-6H) inoculatedwith Phakopsora pachyrhizi. The leaves were photographed at 13- and44-days post inoculation as indicated.

FIGS. 7A-7B are photographs of wild type soybean plants (FIG. 7A) andtransgenic soybean plants expressing FIT1 (FIG. 7B).

FIG. 7C is a bar graph of the height of the plants shown in FIG. 9A andFIG. 9B 24 days after planting. The error bars indicate the standarddeviation of the plant height from the individual plants (n>8).

FIG. 8 depicts an alignment between the FIT1 alleles from Vignaunguiculata (VuFIT1), Phaseolus lunatus (PlFIT1), Abrus precatorius(ApFIT1), and the N gene, which gives TMV resistance. The LRR domain ispoorly conserved between the FIT1 alleles and the N gene.

DETAILED DESCRIPTION Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques and/or substitutionsof equivalent techniques that would be apparent to one of skill in theart.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. For example, the phrase “a cell” refers to one or morecells, and in some embodiments can refer to a tissue and/or an organ.Similarly, the phrase “at least one”, when employed herein to refer toan entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, includingbut not limited to all whole number values between 1 and 100 as well aswhole numbers greater than 100.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” The term “about,” as used herein when referring to ameasurable value such as an amount of mass, weight, time, volume,concentration or percentage is meant to encompass variations of ±10%from the specified amount, as such variations are appropriate to performthe disclosed methods and/or employ the disclosed compositions, nucleicacids, polypeptides, etc. Accordingly, unless indicated to the contrary,the numerical parameters set forth in this specification and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently disclosed subjectmatter.

As used herein, the term “and/or” when used in the context of a list ofentities, refers to the entities being present singly or in combination.Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, andD individually, but also includes any and all combinations andsubcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC,ABD, and BCD). In some embodiments, one or more of the elements to whichthe “and/or” refers can also individually be present in single ormultiple occurrences in the combinations(s) and/or subcombination(s).

As used herein, the term “plant” can refer to any living organismbelonging to the kingdom Plantae (i.e., any genus/species in the PlantKingdom), to a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to any ofwhole plants, plant components or organs (e.g., leaves, stems, roots,etc.), plant tissues, seeds and/or plant cells.

A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant. Thus, the term “plantcell” includes without limitation cells within seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, shoots,gametophytes, sporophytes, pollen, and microspores.

The phrase “plant part” refers to a part of a plant, including singlecells and cell tissues such as plant cells that are intact in plants,cell clumps, and tissue cultures from which plants can be regenerated.Examples of plant parts include, but are not limited to, single cellsand tissues from pollen, ovules, leaves, embryos, roots, root tips,anthers, flowers, fruits, stems, shoots, and seeds; as well as scions,rootstocks, protoplasts, calli, and the like.

As used herein, the term “resistant”, or “resistance”, describes aplant, line or variety that shows fewer or reduced symptoms to a bioticpest or pathogen than a susceptible (or more susceptible) plant, line orvariety to that biotic pest or pathogen. This term is also applied toplants that show no symptoms, and may also be referred to as“high/standard resistance”.

As used herein, the term “tolerant” or “tolerance” describes a plant,line, or variety that that shows some symptoms to a biotic pest orpathogen, but that are still able to produce marketable product with anacceptable yield. These lines may also be referred to as having“moderate/intermediate resistance”. Tolerant and moderate/intermediateresistant plant types restrict the growth and development of thespecified pest or pathogen, but exhibit a greater range of symptoms ordamage compared to plant types with high resistance. Plant types withintermediate resistance will show less severe symptoms than susceptibleplant varieties, when grown under similar field conditions and pathogenpressure. A “tolerant” plant may also indicate a phenotype of a plantwherein disease-symptoms remain absent upon exposure of said plant to aninfective dosage of pathogen, whereby the presence of a systemic orlocal pathogen infection, pathogen multiplication, at least the presenceof pathogen genomic sequences in cells of said plant and/or genomicintegration thereof can be established. Tolerant plants are thereforeresistant for symptom expression but symptomless carriers of thepathogen. Sometimes, pathogen sequences may be present or even multiplyin plants without causing disease symptoms. This phenomenon is alsoknown as “latent infection”. In latent infections, the pathogen mayexist in a truly latent non-infectious occult form, possibly as anintegrated genome or an episomal agent (so that pathogen protein cannotbe found in the cytoplasm, while PCR protocols may indicate the presentof pathogen nucleic acid sequences) or as an infectious and continuouslyreplicating agent. A reactivated pathogen may spread and initiate anepidemic among susceptible contacts. The presence of a “latentinfection” is indistinguishable from the presence of a “tolerant”phenotype in a plant.

Methods of evaluating resistance are well known to one skilled in theart. Such evaluation may be performed by visual observation of a plantor a plant part (e.g., leaves, roots, flowers, fruits et. al) indetermining the severity of symptoms. For example, when each plant isgiven a resistance score on a scale of 1 to 5 based on the severity ofthe reaction or symptoms, with 1 being the resistance score applied tothe most resistant plants (e.g., no symptoms, or with the leastsymptoms), and 5 the score applied to the plants with the most severesymptoms, then a line is rated as being resistant when at least 75% ofthe plants have a resistance score at a 1, 2, or 3 level, whilesusceptible lines are those having more than 25% of the plants scoringat a 4 or 5 level. If a more detailed visual evaluation is possible,then one can use a scale from 1 to 10 so as to broaden out the range ofscores and thereby hopefully provide a greater scoring spread among theplants being evaluated. Additional methods for evaluating resistance arewell known in the art (see for example,jircas.go.jp/sites/default/files/publication/manual_guideline/manual_guideline-_-_73.pdfavailable on the world wide web).

In addition to such visual evaluations, disease evaluations can beperformed by determining the pathogen bio-density in a plant or plantpart using electron and/or light microscopy and/or through molecularbiological methods, such as protein quantification (e.g., ELISA,measuring pathogen protein density) and/or nucleic acid quantification(e.g., RT-PCR, measuring pathogen RNA density). Another method relies onquantifying the spores produced by the pathogen, which can be quantifiedusing a hemacytometer and evaluated per uredinium, per leaf area, or perleaf.

As used herein, the term “susceptible” is used herein to refer to aplant that is unable to prevent entry of the pathogen into the plantand/or slow multiplication and systemic spread of the pathogen,resulting in disease symptoms. The term “susceptible” is thereforeequivalent to “non-resistant”.

As used herein, the term “homologous” or “homolog” is used as it isknown in the art and refers to related sequences that share a commonancestor. The term “homolog” is sometimes used to apply to therelationship between genes separated by the event of speciation(“ortholog”) or to the relationship between genes separated by the eventof genetic duplication within the same species (“paralog”). Homology canbe determined using software programs readily available in the art, suchas those discussed in Current Protocols in Molecular Biology (F. M.Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71.

As used herein, the term “allele” is used both as it is known in the artas one of two or more versions of a gene or peptide, and also to referto synthetic variants of a gene or peptide containing one or morechanges from the native sequence.

As used herein, the term “functional” used in the context of a homologmeans that the homolog has the same or very similar function. Forexample, a functional homolog of FIT1 would recognize an AvrFIT1effector protein. A “nonfunctional FIT1 homolog” would not recognizeAvrFIT1, though it may still be functional in that it is able recognizeother effector proteins.

As used herein, the term “sequence identity” refers to the presence ofidentical nucleotides or amino acids at corresponding positions of twosequences. Readily available sequence comparison and multiple sequencealignment algorithms are, respectively, the Basic Local Alignment SearchTool (BLAST®) and ClustalW/ClustalW2/Clustal Omega programs available onthe Internet (e.g., the website of the EMBL-EBI). Some alignmentprograms are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGNPlus (Scientific and Educational Software, Pennsylvania). Othernon-limiting alignment programs include Sequencher (Gene Codes, AnnArbor, Mich.), AlignX, and Vector NTI (Invitrogen, Carlsbad, Calif.).Other suitable programs include, but are not limited to, GAP, BestFit,Plot Similarity, and FASTA, which are part of the Accelrys GCG Packageavailable from Accelrys, Inc. of San Diego, Calif., United States ofAmerica. See also Smith & Waterman, 1981; Needleman & Wunsch, 1970;Pearson & Lipman, 1988; Ausubel et al., 1988; and Sambrook & Russell,2001. Unless otherwise noted, alignments disclosed herein utilizedClustal Omega.

As used herein, the phrases “DNA construct”, “expression cassette”,“chimeric construct”, “construct”, and “recombinant DNA construct” areused interchangeably herein. A recombinant DNA construct comprises anartificial combination of nucleic acid fragments, e.g., regulatory andcoding sequences that are not found together in nature. For example, aconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such construct may be used byitself or may be used in conjunction with a vector. If a vector is usedthen the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The vector may be a viralvector that is suitable as a delivery vehicle for delivery of thenucleic acid, or mutant thereof, to a cell, or the vector may be anon-viral vector which is suitable for the same purpose. Examples ofviral and non-viral vectors for delivery of DNA to cells and tissues arewell known in the art and are described, for example, in Ma et al.(1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746).

As used herein “cisgene” refers to a gene from the same species, or aspecies closely related enough to be conventionally bred. “Transgene”refers to a gene from a different species, and may also be referred toas “heterologous” (an amino acid or a nucleic acid sequence which is notnaturally found in the particular organism). Both transgenes andheterologous sequences would be considered “exogenous” as referring to asubstance coming from some source other than its native source.

The term “operably linked” refers to the juxtaposition of two or morecomponents (such as sequence elements) having a functional relationship.For example, the sequential arrangement of the promoter polynucleotidewith a further oligo- or polynucleotide, resulting in transcription ofthe further polynucleotide.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Insome embodiments, the promoter sequence consists of proximal and moredistal upstream elements, the latter elements often referred to asenhancers. Accordingly, an “enhancer” is a DNA sequence that canstimulate promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter.

As used herein, “selectable marker” is a nucleic acid segment thatallows one to select for a molecule (e.g., a plasmid) or a cell thatcontains it, often under particular conditions. These markers can encodean activity, such as, but not limited to, production of RNA, peptide, orprotein, or can provide a binding site for RNA, peptides, proteins,inorganic and organic compounds or compositions and the like.

The following description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed disclosures, or that any publication specifically orimplicitly referenced is prior art.

Overview

The present disclosure provides an isolated, recombinant, or syntheticpolynucleotide comprising a FIT1 protein, and homologs, fragments, andvariations thereof. The disclosure further relates to plants, plantparts, and plant cells that have been transformed with thesepolynucleotides, and exhibit resistance or tolerance to a plantpathogen, such as Phakopsora pachyrhizi. The disclosure further relatesto methods of identifying pathogen resistant genes, and methods ofgenetically engineering a pathogen resistance or tolerance trait in aplant, plant part, or plant cell, comprising targeted gene editing of ahomolog (such as FIT1), and plants produced therefrom.

Plant Pathogens

There are more than 6000 species of rust fungi, including for example,Phakopsora pachyrhizi, Puccinia spp., Uromyces appendiculatus, andHemileia vastatrix, that infect a wide range of important crops andornamental varieties. Some examples of varieties that may be infectedinclude, but are not limited to, Avena sativa (oats), Vicia faba (broadbeans), Coffea arabica (coffee), Chrysanthemum, Cydonia (quince),Fuchsia spp. (garlic), Hordeum vulgare (barley), Juniperus virginiana(red cedar), Juniperus communis (juniper), Allium ampeloprasum (leek),Malta spp. (apple), Mentha piperita (peppermint), Mespilus (medlar),Onion, Pelargonium, Primula vulgaris (primrose), Pyrus (pear), Rosa spp.(roses), Triticum spp. (wheat), Secale cereale (rye), Vitis vinifera(grape), Saccharum spp. (sugarcane) and Glycine max (soybean).

In soybean, rust pathogen infections have been reported in SouthAmerica, Africa, Australia, and Asia, and were first reported on soybeancrops in the southern United States in 2004. Asian soybean rust (ASR),caused by Phakopsora pachyrhizi, spreads quickly and can lead tosignificant yield loss. Initial symptoms of ASR include yellowdiscoloration on the upper surfaces of foliage, followed by tan orreddish-brown lesions on the undersides of the leaves and sometimes onpetioles, stems or pods. Blisters develop within the lesions, whichbreak open and release spores. Soybean plants infected with ASR willexhibit reduced pod production and can result in a yield loss of greaterthan 50%. Another disease, New World soybean rust, caused by Phakopsorameibomiae, is generally not as harmful as ASR. P. meibomiae has not yetbeen reported in the U.S.

Successful infection of rust fungi relies on the secretion of effectorproteins with functions that facilitate host colonization. The effectorproteins suppress plant immunity and manipulate the host metabolism tobenefit the pathogen (Jones and Dangl, 2006). AvrFIT1 is one sucheffector protein secreted by Phakopsora pachyrhizi that causes ASR.AvrFIT1 is present in several sequenced Phakopsora pachyrhizi strainsisolated from various locations across the world, including Brazil andNorth America. A phylogenetic tree of close homologs of AvrFIT1 revealsthat there are two similar copies of AvrFIT1 in Phakopsora pachyrhizi(FIG. 1 ), (PpAvrFIT1a and PpAvrFIT1b), both of which are recognized byFIT1 in transient assays. AvrFIT1 is also conserved in many other fungalspecies. Thai1, LA04-1 and MG2006 are three strains of Phakopsorapachyrhizi that all contain recognized alleles of AvrFIT1 (Elmore etal., 2020; Link et al., 2014, and information on Phakopsora pachyrhizifrom the genome portal of the Department of Energy Joint GenomeInstitute available on the worldwide web.). Phakopsora pachyrhizi BR isan unspecified Brazilian population which also contains recognizedalleles of AvrFIT1 (de Carvalho et al., 2017). AvrFIT1 is present inspecies of rust pathogens that cause disease on poplar (Melampsoralarici-populina), cereals (Puccinia spp.) and other plants. Putativeorthologs of AvrFIT1 are also present in more distantly related fungalspecies including both pathogenic and non-pathogenic fungi. For example,AvrFIT1 is present in Rhizoctonia solani, a non-rust pathogen that canbe problematic for herbaceous plants, causing diseases such as collarrot, root rot, damping off, and wire stem.

AvrFIT1 is a putative peptidyl prolyl isomerase and is predicted todisrupt or perturb the function of one or more plant host proteins.While not wishing to be bound by any particular theory, it is possiblethat AvrFIT1 could be modifying an unknown plant protein, possibly aconserved component of the plant immune system, and FIT1 could be“guarding” the unknown protein and be activated if the protein ismodified by AvrFIT1. There are many examples of NLR receptors actingthis way in the literature. Pathogen effectors often convergently evolveto target the same or similar plant host proteins to promote virulence.There are many examples of two different pathogen effector proteinsevolving to target the same plant protein independently. Thus, it ispossible that another pathogen might have an effector protein that isunrelated to AvrFIT1 but acts on the same protein as AvrFIT1 andtherefore is also capable of being perceived by FIT1. By guardingagainst proteins with a similar activity or molecular target as AvrFIT1,FIT1 can mediate resistance against pathogens which don't have a closehomolog of AvrFIT1 but do have a protein which has evolved to have asimilar activity or molecular target as FIT1. Such proteins may bepresent in a diverse range of plant pathogens including but not limitedto fungi, oomycetes, bacteria, nematodes, or viruses.

Plant Resistance

Plants have, in some cases, evolved immunity in which resistance geneproducts recognize the activity of specific effectors resulting ineffector-trigger immunity (ETI) (Jones and Dangl, 2006). ETI leads torobust defenses, such as the hypersensitive response (HR), which is aform of programmed cell death that results in the formation of alocalized lesion that inhibits pathogen growth at the initial infectionsite (Dodds and Rathjen, 2010). If the plant has an immune receptorcapable of recognizing the pathogen effector protein, the effectorprotein activates a strong immune response conferring immunity. Theperception of intracellular pathogen effector proteins in plants isfrequently mediated by proteins from a large gene family known as thenucleotide binding, leucine-rich repeat (NLR) proteins (Jones et al.,2016).

Plant disease resistance traits are often encoded by NLR genes. NLRgenes can be incorporated into a susceptible crop variety to conferresistance through a variety of methods including introgressionbreeding, transformation or genome editing. A typical plant has hundredsof NLR immune receptor genes (Jones et al., 2016). These genes aretypically expressed at relatively low levels with the NLR proteinspassively surveilling for the presence of cognate effector proteins frominvading pathogens. Prior to activation, the NLR proteins haveessentially no impact on plant metabolism or growth. Upon activation bya cognate ligand, typically a pathogen effector protein or a proteinsubstrate of an effector, the NLR protein initiates a signalling cascadethat activates endogenous plant defense pathways to inhibit pathogengrowth. Using NLRs is a natural, safe, and environmentally sustainablemechanism to develop disease-resistant crop varieties to improve plantyields and reduce the need for chemical controls.

FIT1

FIT1 is a plant Toll-like interleukin-1 receptor (TIR) nucleotidebinding leucine rich repeat (NLR) immune receptor protein discovered byApplicants. It was identified from Vigna unguiculata (cowpea) and isresponsible for AvrFIT1 recognition. As shown in FIG. 2 , expression ofFIT1 correlates with resistance to ASR. Accessions of Vigna radiata, (PI378026), Phaseolus lunatus (PI 180324), and Vigna unguiculata (LG104,Baker Creek Heirloom Seeds) which contain functional copies of FIT1genes show strong resistance to Phakopsora pachyrhizi. In contrast,accessions of Glycine max (Williams 82), Pachyrhizus erosus (AB105,Baker Creek Heirloom Seeds), and Glycine peratosa (PI 583964), which alllack functional FIT1, are highly susceptible to Phakopsora pachyrhizi asobserved by large disease lesions and spore production. These resultsindicate a correlation between the presence of FIT1 and resistance toPhakopsora pachyrhizi and suggest that expression of FIT1 in plants canconfer disease resistance. Additionally, the widespread distribution ofAvrFIT1 discussed above suggests that FIT1 can confer disease resistanceagainst a broad range of pathogenic species.

Thus, an embodiment of the present disclosure provides an isolated,recombinant, or synthetic polynucleotide comprising a nucleic acidsequence encoding a FIT1 protein, wherein the protein is selected fromthe group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,functional homologs, and/or fragments and variations thereof. In somecases, the functional FIT1 homolog shares at least about 70% identity toSEQ ID NO: 2 and recognizes an effector protein secreted by a plantpathogen. In some cases, the functional FIT1 homolog recognizes at leastone of AvrFIT1a and AvrFIT1b. In some cases, the functional FIT1 homologshares at least about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%,about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about99.7%, about 99.8%, or about 99.9% identity to SEQ ID NO: 2.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 4.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 6.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 8.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 10.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 12

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 14.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 16.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 18.

In some cases, the functional FIT1 homolog shares at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 20.

In some aspects, the isolated, recombinant, or synthetic polynucleotidecomprises a nucleic acid sequence encoding SEQ ID NO: 2, or an aminoacid sequence at least 90% identical thereto. In some aspects, theisolated, recombinant, or synthetic polynucleotide comprises a nucleicacid sequence encoding SEQ ID NO: 4, or an amino acid sequence at least90% identical thereto. In some aspects, the isolated, recombinant, orsynthetic polynucleotide comprises a nucleic acid sequence encoding SEQID NO: 6, or an amino acid sequence at least 90% identical thereto. Insome aspects, the isolated, recombinant, or synthetic polynucleotidecomprises a nucleic acid sequence encoding SEQ ID NO: 8, or an aminoacid sequence at least 90% identical thereto. In some aspects, theisolated, recombinant, or synthetic polynucleotide comprises a nucleicacid sequence encoding SEQ ID NO: 10, or an amino acid sequence at least90% identical thereto. In some aspects, the isolated, recombinant, orsynthetic polynucleotide comprises a nucleic acid sequence encoding SEQID NO: 12, or an amino acid sequence at least 90% identical thereto. Insome aspects, the isolated, recombinant, or synthetic polynucleotidecomprises a nucleic acid sequence encoding SEQ ID NO: 14, or an aminoacid sequence at least 90% identical thereto. In some aspects, theisolated, recombinant, or synthetic polynucleotide comprises a nucleicacid sequence encoding SEQ ID NO: 16, or an amino acid sequence at least90% identical thereto. In some aspects, the isolated, recombinant, orsynthetic polynucleotide comprises a nucleic acid sequence encoding SEQID NO: 18, or an amino acid sequence at least 90% identical thereto. Insome aspects, the isolated, recombinant, or synthetic polynucleotidecomprises a nucleic acid sequence encoding SEQ ID NO: 20, or an aminoacid sequence at least 90% identical thereto.

In another embodiment, the disclosure relates to a transgenic plant,plant part, or cell having resistance or tolerance to at least one plantpathogen, wherein the resistance or tolerance is conferred by apolynucleotide encoding at least one of the functional FIT1 homologsdisclosed herein.

In another embodiment, the present disclosure provides an isolated,recombinant, or synthetic polynucleotide, wherein the polynucleotidecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 complements thereof,fragments thereof, and sequences at least 70% identical thereto, whereinsaid sequences encode a functional FIT1 protein. In some cases, thepolynucleotide shares at least about 70%, at least about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%,about 99.6%, about 99.7%, about 99.8%, or about 99.9% identity to SEQ IDNO: 1. In some embodiments, the disclosure relates to genetic constructscomprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 3. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 5. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 7. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 9. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 11. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 13. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 15. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 17. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

In some cases, the polynucleotide shares at least about 70%, at leastabout 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, about 79%, 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about99.9% identity to SEQ ID NO: 19. In some embodiments, the disclosurerelates to genetic constructs comprising these sequences.

The disclosure also encompasses variants and fragments of proteins of anamino acid sequence encoded by the nucleic acid sequences of FIT1,orthologs of FIT1 and/or paralogs of FIT1. The variants may containalterations in the amino acid sequences of the constituent proteins. Theterm “variant” with respect to a polypeptide refers to an amino acidsequence that is altered by one or more amino acids with respect to areference sequence. The variant can have “conservative” changes, or“nonconservative” changes, e.g., analogous minor variations can alsoinclude amino acid deletions or insertions, or both.

Functional fragments and variants of a polypeptide include thosefragments and variants that maintain one or more functions or domains ofthe parent polypeptide. As used herein, a protein domain is a distinctfunctional and/or structural unit in a protein, and are usuallyresponsible for a particular function or interaction. It is recognizedthat the gene or cDNA encoding a polypeptide can be considerably mutatedwithout materially altering one or more of the polypeptide's functionsand/or domains. First, the genetic code is well-known to be degenerate,and thus different codons encode the same amino acids. Second, evenwhere an amino acid substitution is introduced, the mutation can beconservative and have no material impact on the essential function(s) ofa protein. See, e.g., Stryer Biochemistry 3^(rd) Ed., 1988. Third, partof a polypeptide chain can be deleted without impairing or eliminatingall of its functions. Fourth, insertions or additions can be made in thepolypeptide chain for example, adding epitope tags, without impairing oreliminating its functions (Ausubel et al. J. Immunol. 159(5): 2502-12,1997). Other modifications that can be made without materially impairingone or more functions of a polypeptide can include, for example, in vivoor in vitro chemical and biochemical modifications or the incorporationof unusual amino acids. Such modifications include, but are not limitedto, for example, acetylation, carboxylation, phosphorylation,glycosylation, ubiquination, labelling, e.g., with radionucleotides, andvarious enzymatic modifications, as will be readily appreciated by thosewell skilled in the art. A variety of methods for labellingpolypeptides, and labels useful for such purposes, are well known in theart, and include radioactive isotopes such as ³²P, ligands which bind toor are bound by labelled specific binding partners (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and anti-ligands.Functional fragments and variants can be of varying length. For example,some fragments have at least 10, 25, 50, 75, 100, 200, or even moreamino acid residues. These mutations can be natural or purposelychanged. In some embodiments, mutations containing alterations thatproduce silent substitutions, additions, or deletions, but do not alterthe properties or activities of the proteins or how the proteins aremade are an embodiment of the present disclosure.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions.Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Furtherinformation about conservative substitutions can be found, for instance,in Ben Bassat et al. (J. Bacteriol., 169:751-757, 1987), O'Regan et al.(Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Sci., 3:240-247,1994), Hochuli et al. (Bio/Technology, 6:1321-1325, 1988) and in widelyused textbooks of genetics and molecular biology. The Blosum matricesare commonly used for determining the relatedness of polypeptidesequences. The Blosum matrices were created using a large database oftrusted alignments (the BLOCKS database), in which pairwise sequencealignments related by less than some threshold percentage identity werecounted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992). A threshold of 90% identity was used for the highly conservedtarget frequencies of the BLOSUM90 matrix. A threshold of 65% identitywas used for the BLOSUM65 matrix. Scores of zero and above in the Blosummatrices are considered “conservative substitutions” at the percentageidentity selected. The following table 1 shows exemplary conservativeamino acid substitutions.

TABLE 1 Very Highly Conserved Conserved Highly - SubstitutionsSubstitutions Orignal Conserved (from the (from the ResidueSubstitutions Blosum90 Matrix) Blosum65 Matrix) Ala Ser Gly, Ser, ThrCys, Gly, Ser, Thr, Val Arg Lys Gln, His, Lys Asn, Gln, Glu, His, LysAsn Gln; His Asp, Gln, His, Lys, Arg, Asp, Gln, Glu, His, Ser, Thr Lys,Ser, Thr Asp Glu Asn, Glu Asn, Gln, Glu, Ser Cys Ser None Ala Gln AsnArg, Asn, Glu, His, Arg, Asn, Asp, Glu, His, Lys, Met Lys, Met, Ser GluAsp Asp, Gln, Lys Arg, Asn, Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, SerHis Asn; Gln Arg, Asn, Gln, Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; ValLeu, Met, Val Leu, Met, Phe, Val Leu Ile; Val Ile, Met, Phe, Val Ile,Met, Phe, Val Lys Arg; Gln; Glu Arg, Asn, Gln, Glu Arg, Asn, Gln, Glu,Ser, Met Leu; Ile Gln, Ile, Leu, Val Gln, Ile, Leu, Phe, Val Phe Met;Leu; Tyr Leu, Trp, Tyr Ile, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, ThrAla, Asn, Asp, Gln, Glu, Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala, Asn,Ser, Val Trp Tyr Phe, Tyr Phe, Tyr Tyr Trp; Phe His, Phe, Trp His, Phe,Trp Val Ile; Leu Ile, Leu, Met Ala, Ile, Leu, Met, Thr

Codon Optimization

In some cases, variants may differ from the disclosed sequences byalteration of the coding region to fit the codon usage bias of theparticular organism into which the molecule is to be introduced. Inother cases, the coding region may be altered by taking advantage of thedegeneracy of the genetic code to alter the coding sequence such that,while the nucleotide sequence is substantially altered, it neverthelessencodes a protein having an amino acid sequence substantially similar tothe disclosed amino acid sequences of FIT1, orthologs of FIT1 and/orparalogs of FIT1, and/or fragments and variations thereof.

Protein expression is governed by a host of factors including those thataffect transcription, mRNA processing, and stability and initiation oftranslation. Optimization can thus address any of a number of sequencefeatures of any particular gene. Translation may be paused due to thepresence of codons in the polynucleotide of interest that are rarelyused in the host organism, and this may have a negative effect onprotein translation due to their scarcity in the available tRNA pool.Specifically, it can result in reduced protein expression.

Alternate translational initiation also can result in reducedheterologous protein expression. Alternate translational initiation caninclude a synthetic polynucleotide sequence inadvertently containingmotifs capable of functioning as a ribosome binding site (RBS). Thesesites can result in initiating translation of a truncated protein from agene-internal site. One method of reducing the possibility of producinga truncated protein includes eliminating putative internal RBS sequencesfrom an optimized polynucleotide sequence.

Repeat-induced polymerase slippage can result in reduced heterologousprotein expression. Repeat-induced polymerase slippage involvesnucleotide sequence repeats that have been shown to cause slippage orstuttering of DNA polymerase which can result in frameshift mutations.Such repeats can also cause slippage of RNA polymerase. In an organismwith a high G+C content bias, there can be a higher degree of repeatscomposed of G or C nucleotide repeats. Therefore, one method of reducingthe possibility of inducing RNA polymerase slippage, includes alteringextended repeats of G or C nucleotides.

Interfering secondary structures also can result in reduced heterologousprotein expression. Secondary structures can sequester the RBS sequenceor initiation codon and have been correlated to a reduction in proteinexpression. Stemloop structures can also be involved in transcriptionalpausing and attenuation. An optimized polynucleotide sequence cancontain minimal secondary structures in the RBS and gene coding regionsof the nucleotide sequence to allow for improved transcription andtranslation.

The optimization process can begin, for example, by identifying thedesired amino acid sequence to be expressed by the host. From the aminoacid sequence, a candidate polynucleotide or DNA sequence can bedesigned. During the design of the synthetic DNA sequence, the frequencyof codon usage can be compared to the codon usage of the host expressionorganism and rare host codons can be removed from the syntheticsequence. Additionally, the synthetic candidate DNA sequence can bemodified in order to remove undesirable enzyme restriction sites and addor remove any desired signal sequences, linkers or untranslated regions.The synthetic DNA sequence can be analyzed for the presence of secondarystructure that may interfere with the translation process, such as G/Crepeats and stem-loop structures.

Optimized coding sequences containing codons preferred by a particularhost can be prepared, for example, to increase the rate of translationor to produce recombinant RNA transcripts having desirable properties,such as a longer half-life, as compared with transcripts produced from anon-optimized sequence.

Functional Fragments, Chimeric, and Synthetic Polypeptides

In some cases, functional fragments derived from FIT1 orthologs of thepresent disclosure can still confer resistance to pathogens whenexpressed in a plant. In some cases, the functional fragments containone or more conserved regions shared by two or more FIT1 orthologs.

In some cases, functional chimeric or synthetic polypeptides derivedfrom the FIT1 orthologs of the present disclosure are provided. Thefunctional chimeric or synthetic polypeptides can still conferresistance to pathogens when expressed in a plant. In some cases, thefunctional chimeric or synthetic polypeptides contain one or moreconserved regions shared by two or more FIT1 orthologs.

DNA Constructs

In some embodiments, the disclosure relates to a DNA constructcomprising at least one FIT1 sequence disclosed herein. In some cases,the FIT1 sequence is a polynucleotide comprising a nucleic acid sequenceencoding a FIT1 protein, wherein the protein is selected from the groupconsisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,functional homologs, and/or fragments and variations thereof. In somecases, the FIT1 protein shares at least about 70% identity to SEQ ID NO:2. In some cases, the FIT1 sequence is selected from SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19 complements thereof, fragments thereof, andsequences at least 70% identical thereto. In some cases, two or moreFIT1 sequences are stacked to increase pathogen resistance in a plant.In some cases, at least one FIT1 sequence is stacked with anotherpathogen resistance gene.

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make DNA constructs for usein the present disclosure. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Lastly,plant promoters such as prolifera promoter, fruit specific promoters,Ap3 promoter, heat shock promoters, seed specific promoters, etc. canalso be used.

Either a gamete-specific promoter, a constitutive promoter (such as theCaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato), or an inducible promoter is typically ligated tothe protein or antisense encoding region using standard techniques knownin the art. The DNA construct may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

Thus, for expression in plants, the DNA construct will typicallycontain, in addition to the protein sequence, a plant promoter region, atranscription initiation site and a transcription termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the expressionunit are typically included to allow for easy insertion into apre-existing vector.

In the construction of heterologous promoter/gene of interest orantisense combinations, the promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the gene toprovide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. If the mRNA encoded by the gene is to beefficiently processed, DNA sequences which direct polyadenylation of theRNA are also commonly added to the vector construct. Polyadenylationsequences include, but are not limited to the Agrobacterium octopinesynthase signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopalinesynthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573(1982)). The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. One or more expression units may be included inthe same vector.

Selection

A DNA construct will typically contain a selectable marker geneexpression unit by which transformed plant cells can be identified inculture. Usually, the marker gene will encode resistance to anantibiotic, such as G418, hygromycin, bleomycin, kanamycin, orgentamicin or to an herbicide, such as glyphosate (Round-Up) orglufosinate (BASTA) or atrazine. Replication sequences, of bacterial orviral origin, are generally also included to allow the vector to becloned in a bacterial or phage host; preferably a broad host range forprokaryotic origin of replication is included. A selectable marker forbacteria may also be included to allow selection of bacterial cellsbearing the desired construct. Suitable prokaryotic selectable markersinclude resistance to antibiotics such as ampicillin, kanamycin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art. For instance, in thecase of Agrobacterium transformations, T-DNA sequences will also beincluded for subsequent transfer to plant chromosomes.

For positive selection, for example, a foreign gene is supplied to aplant cell that allows it to utilize a substrate present in the mediumthat it otherwise could not use, such as mannose or xylose (for example,refer U.S. Pat. Nos. 5,767,378; 5,994,629). More typically, however,negative selection is used because it is more efficient, utilizingselective agents such as herbicides or antibiotics that either kill orinhibit the growth of non-transformed plant cells and reducing thepossibility of chimeras. Resistance genes that are effective againstnegative selective agents are provided on the introduced foreign DNAused for the plant transformation. For example, one of the most popularselective agents used is the antibiotic kanamycin, together with theresistance gene neomycin phosphotransferase (nptII), which confersresistance to kanamycin and related antibiotics (see, for example,Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature304:184-187 (1983)). However, many different antibiotics and antibioticresistance genes can be used for transformation purposes (refer U.S.Pat. Nos. 5,034,322, 6,174,724 and 6,255,560). In addition, severalherbicides and herbicide resistance genes have been used fortransformation purposes, including the bar gene, which confersresistance to the herbicide phosphinothricin (White et al., Nucl AcidsRes 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625-631(1990),U.S. Pat. Nos. 4,795,855, 5,378,824 and 6,107,549). In addition, thedhfr gene, which confers resistance to the anticancer agentmethotrexate, has been used for selection (Bourouis et al., EMBO J.2(7): 1099-1104 (1983).

Transgenic Plants Comprising Sequences Disclosed Herein

In one embodiment, the present disclosure relates to a transgenic plant,plant part, or plant cell, wherein the transgene comprises at least onepolynucleotide coding for FIT1, orthologs of FIT1 and/or paralogs ofFIT1, and/or fragments and variations thereof, and exhibit resistance ortolerance to a pathogen. In some cases, the polynucleotide encodes aprotein selected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, and proteins at least 90% identical theretofunctional homologs thereof. In some cases, the polynucleotide comprisesa nucleic acid sequence selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 complements thereof, fragmentsthereof, and sequences at least 70% identical thereto. In some cases,the pathogen is a fungus. In some cases, the fungus is from the orderCantharellales or Pucciniales. In some cases, the fungal pathogen isRhizoctonia solani, Melampsora spp., Phakopsora pachyrhizi, Phakopsorameibomiae, Phakopsora euvitis, Phakopsora spp., Puccinia spp., Uromycesspp., Austropuccinia spp., Cronartium spp. or Hemileia vastatrix.

In some cases, the plant, plant part, or plant cell is in the subfamilyPapilionoideae. In some cases, the plant, plant part, or plant cell isAlysicarpus spp., Astragalus spp., Baptisia spp., Cajanus spp.,Calopogonium spp., Caragana spp., Centrosema spp., Cologania spp.,Crotalaria spp., Desmodium spp., Genista spp., Glycine spp., Glycyrrhizaspp., Indigofera spp., Kummerowia spp., Lablab spp., Lathyrus spp.,Lespedeza spp., Lotus spp., Lupinus spp., Macroptilium spp., Macrotylomaspp., Medicago spp., Neonotonia spp., Pachyrhizus spp., Pisum spp.,Phaseolus spp., Pseudovigna spp., Psoralea spp., Robinia spp., Sennaspp., Sesbania spp., Strophostyles spp., Tephrosia spp., Teramnus spp.,Trifolium spp., Vicia spp., Vigna spp., or Voandzeia spp.

In some cases, the plant, plant part, or plant cell is Glycine max, andthe plant, plant part, or plant cell is resistant to Asian Soybean Rustcaused by Phakopsora pachyrhizi.

Methods of producing transgenic plants are well known to those ofordinary skill in the art. Transgenic plants can now be produced by avariety of different transformation methods including, but not limitedto, electroporation; microinjection; microprojectile bombardment, alsoknown as particle acceleration or biolistic bombardment; viral-mediatedtransformation; and Agrobacterium-mediated transformation. See, forexample, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880;5,550,318; 5,641,664; 5,736,369 and 5,736,369; International PatentApplication Publication Nos. WO2002/038779 and WO/2009/117555; Lu etal., (Plant Cell Reports, 2008, 27:273-278); Watson et al., RecombinantDNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama etal., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839(1990); Mullins et al., Bio/Tech. 8:833-839 (1990); Hiei et al., PlantMolecular Biology 35:205-218 (1997); Ishida et al., Nature Biotechnology14:745-750 (1996); Zhang et al., Molecular Biotechnology 8:223-231(1997); Ku et al., Nature Biotechnology 17:76-80 (1999); and, Raineri etal., Bio/Tech. 8:33-38 (1990)), each of which is expressly incorporatedherein by reference in their entirety.

Microprojectile bombardment is also known as particle acceleration,biolistic bombardment, and the gene gun (Biolistic® Gene Gun). The genegun is used to shoot pellets that are coated with genes (e.g., fordesired traits) into plant seeds or plant tissues in order to get theplant cells to then express the new genes. The gene gun uses an actualexplosive (.22 caliber blank) to propel the material. Compressed air orsteam may also be used as the propellant. The Biolistic® Gene Gun wasinvented in 1983-1984 at Cornell University by John Sanford, EdwardWolf, and Nelson Allen. It and its registered trademark are now owned byE. I. du Pont de Nemours and Company. Most species of plants can be beentransformed using this method.

The most common method for the introduction of new genetic material intoa plant genome involves the use of living cells of the bacterialpathogen Agrobacterium tumefaciens. Agrobacterium tumefaciens is anaturally occurring bacterium that is capable of inserting its DNA(genetic information) into plants, resulting in a type of injury to theplant known as crown gall. Most species of plants can now be transformedusing this method. There are numerous patents governing Agrobacteriummediated transformation and particular DNA delivery plasmids designedspecifically for use with Agrobacterium—for example, U.S. Pat. No.4,536,475, EP0265556, EP0270822, WO8504899, WO8603516, U.S. Pat. No.5,591,616, EP0604662, EP0672752, WO8603776, WO9209696, WO9419930,WO9967357, U.S. Pat. No. 4,399,216, WO8303259, U.S. Pat. No. 5,731,179,EP068730, WO9516031, U.S. Pat. Nos. 5,693,512, 6,051,757 and EP904362A1.Methods of Agrobacterium-mediated plant transformation that involveusing vectors with no T-DNA are also well known to those skilled in theart and can be used with the methods of the present disclosure. See, forexample, U.S. Pat. No. 7,250,554, which utilizes P-DNA instead of T-DNAin the transformation vector. A transgenic plant formed usingAgrobacterium transformation methods typically contains a single gene onone chromosome, although multiple copies are possible. Such transgenicplants can be referred to as being hemizygous for the added gene, or maybe referred to as an independent segregant, because each transformedplant represents a unique T-DNA integration event (U.S. Pat. No.6,156,953).

Direct plant transformation methods using DNA have also been reported.The first of these to be reported historically is electroporation, whichutilizes an electrical current applied to a solution containing plantcells (M. E. Fromm et al., Nature, 319, 791 (1986); H. Jones et al.,Plant Mol. Biol., 13, 501 (1989) and H. Yang et al., Plant Cell Reports,7, 421 (1988).

Another direct method, called “biolistic bombardment”, uses ultrafineparticles, usually tungsten or gold, that are coated with DNA and thensprayed onto the surface of a plant tissue with sufficient force tocause the particles to penetrate plant cells, including the thick cellwall, membrane and nuclear envelope (U.S. Pat. Nos. 5,204,253,5,015,580).

A third direct method uses fibrous forms of metal or ceramic consistingof sharp, porous or hollow needle-like projections that impale thecells, and also the nuclear envelope of cells. Both silicon carbide andaluminium borate whiskers have been used for plant transformation(Mizuno et al., 2004; Petolino et al., 2000; U.S. Pat. No. 5,302,523U.S. Application Publication No. 2004/0197909) and also for bacterialand animal transformation (Kaepler et al., 1992; Raloff, 1990; Wang,1995).

Examples of viral vectors include, but are not limited to, recombinantplant viruses. Non-limiting examples of plant viruses include,TMV-mediated (transient) transfection into tobacco (Tuipe, T-H et al(1993), J. Virology Meth, 42: 227-239), ssDNA genomes viruses (e.g.,family Geminiviridae), reverse transcribing viruses (e.g., familiesCaulimoviridae, Pseudoviridae, and Metaviridae), dsNRA viruses (e.g.,families Reoviridae and Partitiviridae), (−) ssRNA viruses (e.g.,families Rhabdoviridae and Bunyaviridae), (+) ssRNA viruses (e.g.,families Bromoviridae, Closteroviridae, Comoviridae, Luteoviridae,Potyviridae, Sequiviridae and Tombusviridae) and viroids (e.g., familiesPospiviroldae and Avsunviroidae). Detailed classification information ofplant viruses can be found in Fauquet et al (2008, “Geminivirus straindemarcation and nomenclature”. Archives of Virology 153:783-821,incorporated herein by reference in its entirety), and Khan et al.(Plant viruses as molecular pathogens; Publisher Routledge, 2002, ISBN1560228954, 9781560228950). Examples of non-viral vectors include, butare not limited to, liposomes, polyamine derivatives of DNA, and thelike.

Non-limiting examples of binary vectors suitable for soybean speciestransformation and transformation methods are described by Yi et al.2006 (Transformation of multiple soybean cultivars by infectingcotyledonary-node with Agrobacterium tumefaciens, African Journal ofBiotechnology Vol. 5 (20), pp. 1989-1993, 16 Oct. 2006), Paz et al.,2004 (Assessment of conditions affecting Agrobacterium-mediated soybeantransformation using the cotyledonary node explant, Euphytica 136:167-179, 2004), U.S. Pat. Nos. 5,376,543, 5,416,011, 5,968,830, and5,569,834, or by similar experimental procedures well known to thoseskilled in the art.

Genes can also be introduced in a site directed fashion using homologousrecombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome. Homologous recombination and site-directed integrationin plants are discussed in, for example, U.S. Pat. Nos. 5,451,513;5,501,967 and 5,527,695.

Genetically Engineering a Pathogen Resistance or Tolerance Trait in aPlant, Plant Part, or Plant Cell

An embodiment of the present disclosure teaches a method of geneticallyengineering a pathogen resistance or tolerance trait in a plant, plantpart, or plant cell, comprising: providing a plant species that issusceptible to a pathogen; identifying within the genome of the plantspecies a homolog of FIT1, wherein said homolog is nonfunctional (doesnot mediate AvrFIT1 recognition), and genetically modifying a plant,plant part, or plant cell from the susceptible plant species withtargeted gene editing, wherein said targeted gene editing is directedtowards the nonfunctional FIT1 homolog, and wherein said targeted geneediting restores the function of FIT1 (enables the FIT1 homolog torecognize AvrFIT1) and confers resistance or tolerance to a pathogen. Insome embodiments, the pathogen is a fungus.

As used herein, a “nonfunctional” FIT1 homolog is a homolog that doesnot recognize a pathogen effector protein homolog of AvrFIT1, such asAvrFIT1a and/or AvrFIT1b. FIT1 homologous may identified by any numberof means known in the art. Methods of alignment of sequences forcomparison are well known in the art. Various programs and alignmentalgorithms are described in: Smith and Waterman (Adv. Appl. Math.,2:482, 1981); Needleman and Wunsch (J. Mol. Biol., 48:443, 1970);Pearson and Lipman (Proc. Natl. Acad. Sci., 85:2444, 1988); Higgins andSharp (Gene, 73:237-44, 1988); Higgins and Sharp (CABIOS, 5:151-53,1989); Corpet et al. (Nuc. Acids Res., 16:10881-90, 1988); Huang et al.(Comp. Appls Biosci., 8:155-65, 1992); and Pearson et al. (Meth. Mol.Biol., 24:307-31, 1994). Altschul et al. (Nature Genet., 6:119-29, 1994)presents a detailed consideration of sequence alignment methods andhomology calculations.

Restoring the function of a FIT1 homolog as used herein relates tomodifying the allele such that it restores the recognition of a pathogeneffector protein such as AvrFIT1a and/or AvrFIT1b, and confersresistance or tolerance to a pathogen. Restoring the function of ahomologous gene by way of genetic engineering has been done and is wellknown in the art (see for example, Ivics Z, et al., (1997), “Molecularreconstruction of Sleeping Beauty, a Tc1-like transposon from fish, andits transposition in human cells”, Cell. 91 (4): 501-510, and recently,Suh S, et al., Restoration of visual function in adult mice with aninherited retinal disease via adenine base editing, Nat Biomed Eng(2020) and Sedeek K, et al., Plant genome engineering for targetedimprovement of crop traits, Front. Plant 12 Feb. 2019).

In some embodiments, the targeted gene editing uses an engineered ornatural nuclease selected from the group consisting of homingendonucleases/meganucleases (EMNs), zinc finger nucleases (ZFNs), andtranscription activator-like effector nucleases (TALENs). In someembodiments, the targeted gene editing uses a clustered regularlyinterspaced short palindromic repeats (CRISPR)-Cas nuclease. In someembodiments, the nuclease is selected from the group consisting of Cas9,Cas12, Cas13, CasX, and CasY. The disclosure also relates to plants,plant parts, and plant cells exhibiting resistance or tolerance to apathogen produced by genetic modification of a FIT1 homolog.

Gene Editing Using CRISPR

Targeted gene editing can be done using CRISPR technology (Saunders &Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as select bacteria and archaea, to respond to andeliminate invading genetic material. Ishino, Y., et al. J. Bacteriol.169, 5429-5433 (1987). These repeats were known as early as the 1980s inE. coli, but Barrangou and colleagues demonstrated that S. thermophiluscan acquire resistance against a bacteriophage by integrating a fragmentof a genome of an infectious virus into its CRISPR locus. Barrangou, R.,et al. Science 315, 1709-1712 (2007). Many plants have already beenmodified using the CRISPR system, including soybean (see for example,Han J, et al., Creation of early flowering germplasm of soybean byCRISPR/Cas9 Technology, Front. Plant Sci., 22 Nov. 2019), and many Casgenes have now been characterized and used with the system (see forexample, Wang J, et al., The rapidly advancing Class 2 CRISPR-Castechnologies: A customizable toolbox for molecular manipulations. J CellMol Med. 2020; 24(6):3256-3270).

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:237-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476.

Gene Editing Using TALENs

Transcription activator-like effector nucleases (TALENs) have beensuccessfully used to introduce targeted mutations via repair of doublestranded breaks (DSBs) either through non-homologous end joining (NHEJ),or by homology-directed repair (HDR) and homology-independent repair inthe presence of a donor template. Thus, TALENs are another mechanism fortargeted genome editing in plants. The technique is well known in theart; see for example Malzahn, Aimee et al. “Plant genome editing withTALEN and CRISPR” Cell & Bioscience vol. 7 21. 24 Apr. 2017.

Other Methods of Genome Editing

In addition to CRISPR and TALENs, two other types of engineerednucleases can be used for genome editing: engineered homingendonucleases/meganucleases (EMNs), and zinc finger nucleases (ZFNs).These methods are well known in the art. See for example, Petilino,Joseph F. “Genome editing in plants via designed zinc finger nucleases”In Vitro Cell Dev Biol Plant. 51(1): pp. 1-8 (2015); and Daboussi,Fayza, et al. “Engineering Meganuclease for Precise Plant GenomeModification” in Advances in New Technology for Targeted Modification ofPlant Genomes. Springer Science+Business. pp 21-38 (2015).

Breeding Methods

Once a gene has been introduced into a plant, or a gene has beengenetically modified, that plant can then be used in conventional plantbreeding schemes (e.g., pedigree breeding, single-seed-descent breedingschemes, recurrent selection, backcross breeding) to produce progenywhich also contain the gene or modified trait. Thus, another aspect ofthe present disclosure relates to breeding with, or asexuallypropagating, plants having been transformed with a FIT1 homolog or animmune receptor gene coding for a protein that recognizes AvrFIT1aand/or AvrFIT1b, or plants wherein a FIT1 nonfunctional homolog wasgenetically modified to recognize AvrFIT1a and/or AvrFIT1b, wherein theplants exhibit resistance or tolerance to a pathogen. The disclosurefurther relates to progeny plants produced therefrom.

In some cases, plants or progeny therefrom comprising the gene ormodified trait may further comprise one or more additional desiredtraits. In some cases, the one or more additional desired traits arestacked on the same construct as the gene (for example, the FIT1 genesdisclosed herein). In another case, the one or more additional desiredtraits may be introgressed by conventional breeding.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent. As used herein, backcross breeding is synonymouswith introgression. Plants produced therefrom may be referred to asingle locus converted or single gene converted plants.

A non-limiting example of a backcross breeding protocol would be thefollowing: a) the first generation F₁ produced by the cross of therecurrent parent A by the donor parent B is backcrossed to parent A, b)selection is practiced for the plants having the desired trait of parentB, c) selected plants are self-pollinated to produce a population ofplants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

Examples of desired traits include, but are not limited to, herbicideresistance (such as bar or pat genes), resistance for bacterial, fungal,or viral disease (such as gene I used for BCMV resistance), insectresistance, enhanced nutritional quality (such as 2s albumin gene),industrial usage, agronomic qualities (such as the “persistent greengene”), yield stability, and yield enhancement.

Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease of new cultivars. Similarly, the development of new cultivarsthrough the dihaploid system requires the selection of the cultivarsfollowed by two to five years of testing in replicated plots.

Open-Pollination

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity. Uniformity in such populationsis impossible and trueness-to-type in an open-pollinated variety is astatistical feature of the population as a whole, not a characteristicof individual plants. Thus, the heterogeneity of open-pollinatedpopulations contrasts with the homogeneity (or virtually so) of inbredlines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes for flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement. First, there is the situation in which a population ischanged en masse by a chosen selection procedure. The outcome is animproved population that is indefinitely propagable by random-matingwithin itself in isolation. Second, the synthetic variety attains thesame end result as population improvement but is not itself propagableas such; it has to be reconstructed from parental lines or clones. Theseplant breeding procedures for improving open-pollinated populations arewell known to those skilled in the art and comprehensive reviews ofbreeding procedures routinely used for improving cross-pollinated plantsare provided in numerous texts and articles, including: Allard,Principles of Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds,Principles of Crop Improvement, Longman Group Limited (1979); Hallauerand Miranda, Quantitative Genetics in Maize Breeding, Iowa StateUniversity Press (1981); and, Jensen, Plant Breeding Methodology, JohnWiley & Sons, Inc. (1988). For population improvement methods specificfor soybean see, e.g., J. R. Wilcox, editor (1987) SOYBEANS:Improvement, Production, and Uses, Second Edition, American Society ofAgronomy, Inc., Crop Science Society of America, Inc., and Soil ScienceSociety of America, Inc., publishers, 888 pages.

Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some cases the donor or recipient female parent and the donoror recipient male parent line are planted in the same field or in thesame greenhouse. The inbred male parent can be planted earlier than thefemale parent to ensure adequate pollen supply at the pollination time.Pollination is started when the female parent flower is ready to befertilized. Female flower buds that are ready to open in the followingdays are identified, covered with paper cups or small paper bags thatprevent bee or any other insect from visiting the female flowers, andmarked with any kind of material that can be easily seen the nextmorning. The male flowers of the male parent are collected in the earlymorning before they are open and visited by pollinating insects. Thecovered female flowers of the female parent, which have opened, areun-covered and pollinated with the collected fresh male flowers of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventbees and any other insects visit. The pollinated female flowers are alsomarked. The marked flowers are harvested. In some cases, the male pollenused for fertilization has been previously collected and stored.

Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. More female plants may be planted to allow for agreater production of seed. Insects are placed in the field orgreenhouses for transfer of pollen from the male parent to the femaleflowers of the female parent.

Mass Selection

In mass selection, desirable individual plants are chosen, harvested,and the seed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

Synthetics

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Parents are selected on general combining ability,sometimes by test crosses or toperosses, more generally by polycrosses.Parental seed lines may be deliberately inbred (e.g., by selfing or sibcrossing). However, even if the parents are not deliberately inbred,selection within lines during line maintenance will ensure that someinbreeding occurs. Clonal parents will, of course, remain unchanged andhighly heterozygous.

Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugar beet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Hybrids may be fertile or sterile depending on qualitative and/orquantitative differences in the genomes of the two parents. Heterosis,or hybrid vigor, is usually associated with increased heterozygositythat results in increased vigor of growth, survival, and fertility ofhybrids as compared with the parental lines that were used to form thehybrid. Maximum heterosis is usually achieved by crossing twogenetically different, highly inbred lines.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., Bulk segregant analysis with molecular markers and its use forimproving drought resistance in maize, 1999, Journal of ExperimentalBotany, 50(337): 1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F₂, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to pathogen), and theother from the individuals having reversed phenotype (e.g., susceptibleto pathogen), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs). Markers linked to thephenotype can be identified and used for breeding or QTL mapping.

Gene Pyramiding

The method to combine into a single genotype a series of target genesidentified in different parents is usually referred as gene pyramiding.The first part of a gene pyramiding breeding is called a pedigree and isaimed at cumulating one copy of all target genes in a single genotype(called root genotype). The second part is called the fixation steps andis aimed at fixing the target genes into a homozygous state, that is, toderive the ideal genotype (ideotype) from the root genotype. Genepyramiding can be combined with marker assisted selection (MAS, seeHospital et al., 1992, 1997a, and 1997b, and Moreau et al, 1998) ormarker based recurrent selection (MBRS, see Hospital et al., 2000).

Examples of Additional Desired Traits that May be Stacked with thePathogen Resistance or Tolerance Traits Disclosed Herein

In some cases, multiple FIT1 alleles may be combined in a single plantto increase pathogen resistance. In some cases, one or more FIT1 allelesare combined with additional desired traits. These traits may beintroduced to a plant through conventional breeding methods, stacked onone or more DNA constructs, and/or generated through targetedmutagenesis. Examples of additional desired traits include, but are notlimited to, male sterility, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. Several of these traits are described in, forexample, U.S. Pat. Nos. 5,959,185, 5,973,234, and 5,977,445.

Examples of Plant Species that May be Transformed or Modified, or Serveas a Source of Functional FIT1

The methods disclosed herein may be applied to a wide range of plants.Non-limiting examples of plants which may be transformed or modifiedusing the methods and sequences disclosed herein include, but are notlimited to, corn (Zea mays), Brassica spp. (e.g., Brassica napus,Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), broad beans (Vicia faba), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), quince (Cydonia), sweet potato (Ipomoeabatatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), apple (Malus spp.), medlar (Mespilus), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), pear (Pyrus), cashew(Anacardium occidentale), macadamia (Macadamia integrifolia), almond(Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharumspp.), oats (for example Avena sativa), barley (for example Hordeumvulgare), vegetables and herbs (for example onion, leek, garlicpeppermint), ornamentals (for example, Chrysanthemum, Fuchsia spp.,Pelargonium, Rosa spp. Primula vulgaris), red cedar (Juniperusvirginiana), and conifers (for example juniper (Juniperus communis)).

Examples of plants in the subfamily Papilionoideae include, but are notlimited to, Alysicarpus spp., Astragalus spp., Baptisia spp., Cajanusspp., Calopogonium spp., Caragana spp., Centrosema spp., Cologania spp.,Crotalaria spp., Desmodium spp., Genista spp., Glycine spp., Glycyrrhizaspp., Indigofera spp., Kummerowia spp., Lablab spp., Lathyrus spp.,Lespedeza spp., Lotus spp., Lupinus spp., Macroptilium spp., Macrotylomaspp., Medicago spp., Neonotonia spp., Pachyrhizus spp., Pisum spp.,Phaseolus spp., Pseudovigna spp., Psoralea spp., Robinia spp., Sennaspp., Sesbania spp., Strophostyles spp., Tephrosia spp., Teramnus spp.,Trifolium spp., Vicia spp., Vigna spp., or Voandzeia spp.

Examples of legumes include, but are not limited to, the genus Phaseolus(e.g., French bean, dwarf bean, climbing bean (Phaseolus vulgaris), limabean (Phaseolus lunatus), Tepary bean (Phaseolus acutifolius), runnerbean (Phaseolus coccineus)); the genus Glycine (e.g., Glycine soja,soybeans (Glycine max (L.)); pea (Pisum) (e.g., shelling peas (sometimecalled smooth or round seeded peas; Pisum sativum); marrowfat pea (Pisumsativum), sugar pea (Pisum sativum), also called snow pea, edible-poddedpea or mangetout, (Pisum grander)); peanut (Arachis hypogaea), clover(Trifolium spp.), medick (Medicago), kudzu vine (Pueraria lobata),common lucerne, alfalfa (Medicago sativa), chickpea (Cicer), lentils(Lens culinaris), lupins (Lupinus); vetches (Vicia), field bean, broadbean (Vicia faba), vetchling (Lathyrus) (e.g., chickling pea (Lathyrussativus), heath pea (Lathyrus tuberosus)); genus Vigna (e.g., moth bean(Vigna aconitifolia), adzuki bean (Vigna angularis), urd bean (Vignamungo), mung bean (Vigna radiata), bambara groundnut (Vigna subterrane),rice bean (Vigna umbellata), Vigna vexillata, Vigna unguiculata (alsoknown as asparagus bean, cowpea)); pigeon pea (Cajanus cajan), the genusMacrotyloma (e.g., geocarpa groundnut (Macro tyloma geocarpum), horsebean (Macrotyloma uniflorum; goa bean (Psophocarpus tetragonolobus),African yam bean (Sphenostylis stenocarpa), Egyptian black bean, lablabbean (Lablab purpureus), yam bean (Pachyrhizus erosus), guar bean(Cyamopsis tetragonolobus); and/or the genus Canavalia (e.g., jack bean(Canavalia ensiformis)), sword bean (Canavalia gladiata).

EXAMPLES

The following examples are provided to illustrate further the variousapplications and are not intended to limit the disclosure beyond thelimitations set forth in the appended claims.

Example 1: FIT1 Homologs

As will be understood by one skilled in the art, homologs of FIT1 may befound in any number of species by methods described herein and methodswell known in the art. Examples of homologs of FIT1 identified are shownin FIGS. 3A-3C. FIG. 3A shows a phylogenetic tree of homologs of Vignaunguiculata FIT1 identified by performing a BLAST® search andconstructing a protein alignment and phylogenetic tree of the resultingsequences. This FIG. shows putative FIT1 orthologs in Vigna radiata,Vigna angularis, Phaseolus acutifolius, Phaseolus lunatus, Phaseolusvulgaris, Lablab purpureus, Mucuna pruriens, Cajanus cajan, and Abrusprecatorius. The phylogenetic tree was rooted using paralogs of FIT1that do not function in AvrFIT1 perception.

FIG. 3B shows a protein alignment of the amino acid sequences listed inFIG. 3A, specifically of the Vigna unguiculata allele of FIT1 (VuFIT1)(SEQ ID NO: 2), the Vigna unguiculata close paralog of FIT1 (VuFIT1b)(SEQ ID NO: 22), Vigna angularis allele of FIT1 (VaFIT1) (SEQ ID NO:12), the Vigna radiata allele of FIT1 (VrFIT1) (SEQ ID NO: 4), thePhaseolus acutifolius allele of FIT1 (PaFIT1) (SEQ ID NO: 10), thePhaseolus vulgaris allele of FIT1 (PvFIT1) (SEQ ID NO: 8), the Phaseoluslunatus allele of FIT1 (PlFIT1) (SEQ ID NO: 6), the Lablab purpureusallele of FIT1 (LpFIT1) (SEQ ID NO: 14), the Cajanus cajun allele ofFIT1 (CcFIT1) 1 (SEQ ID NO: 18), the Mucuna pruriens allele of FIT1(MpFIT1) (SEQ ID NO: 16), and the Abrus precatorius allele of FIT1(ApFIT1) (SEQ ID NO: 20).

FIG. 3C shows a phylogenetic tree of FIT1 homologs in Glycine max. G.max (soybean) lacks an ortholog of VuFIT1 but contains many homologs ofVuFIT1 paralogs, which can be identified by BLAST® search. Proteinhomologs of VuFIT1 from soybean were obtained from NCBI and Phytozome,curated for completeness and to remove duplicates, and used to generatethe phylogenetic tree shown in FIG. 3C. The tree was rooted using anoutgroup of distantly related TIR-NLR proteins from non-legumes.

Sequence alignments of FIT1 homologs with functional FIT1 genes (such asVuFIT1) can show what genetic changes could be induced to restorerecognition of AvrFIT, and confer resistance or tolerance to a pathogen.Such targeted genetic editing is well known in the art and alsodescribed herein.

Example 2: Transient Expression of FIT1 Alleles in Leaf Tissue

Leaf tissue from a plant lacking an endogenous FIT1 was transformed withconstructs containing various FIT1 sequences, AvrFIT1a, AvrFIT1b, and/orBs3 using standard transformation technology (an example constructcomprising VuFIT1 is shown in FIG. 4 ). Suspensions containing thedesired expression constructs were infiltrated into the leaf tissue(OD₆₀₀=0.4 total) using needleless syringe and imaged four days postinfiltration. The site of each infiltration is visible by a small punchthrough the leaf.

As shown in FIGS. 5A and 5B, co-expression of AvrFIT1a (SEQ ID NO: 23)with either Vigna unguiculata (VuFIT1) (SEQ ID NO: 27), Phaseoluslunatus (PlFIT1) (SEQ ID NO: 28), Vigna radiata (VrFIT1) (SEQ ID NO:29), Vigna angularis (VaFIT1) (SEQ ID NO: 31), Phaseolus vulgaris(PvFIT1) (SEQ ID NO: 32), Lablab purpureus (LpFIT1) (SEQ ID NO: 33), orAbrus precatoris (ApFIT1) (SEQ ID NO: 34) resulted in a strong celldeath response, indicative of immune activation, but no response wasobserved when the proteins were expressed individually. No immuneresponse was observed when the non-functional paralog VuFIT1b (SEQ IDNO: 30) was expressed individually or with AvrFIT1a.

As shown in FIGS. 5C and 5D, co-expression of AvrFIT1b (SEQ ID NO: 25)with either Phaseolus lunatus (PlFIT1), Vigna radiata (VrFIT1), Vignaangularis (VaFIT1), Phaseolus vulgaris (PvFIT1), or Lablab purpureus(LpFIT1) resulted in a cell death response, indicative of immuneactivation, but no response was observed when the proteins wereexpressed individually.

Expression of the executor Bs3, a positive control for cell deathresponse, triggers a similar response. This demonstrates that FIT1mediates the perception of AvrFIT1, and thus can confer resistance ortolerance to pathogens that secrete the effector protein AvrFIT1.

Example 3: Stable Expression of Vigna unguiculata FIT1 in Glycine max

Soybean plants stably expressing VuFIT1 (SEQ ID NO: 1, example constructshown in FIG. 4 ) were generated and tested for resistance to ASR(Phakopsora pachyrhizi). FIGS. 6A-6D depicts wild type soybean leaveslacking functional FIT1 (top row) and FIGS. 6E-6H show leaves fromsoybean plants expressing VuFIT1 (bottom row). Plants were inoculatedwith Phakopsora pachyrhizi and the leaves were photographed at 13-days(FIGS. 6A-6B and 6E-6F) and 44-days (FIGS. 6C-6D and 6G-6H) postinoculation. The wild type soybean leaves showed susceptibility toPhakopsora as seen by the development of large lesions and many fungalspores. However, soybean expressing VuFIT1 showed strong resistance tothe pathogen (FIGS. 6E-6H).

Transgenic expression of VuFIT1 in soybean did not affect plant growthor morphology. Photographs of wild type soybean plants (FIG. 7A) andtransgenic soybean plants expressing VuFIT1 (FIG. 7B) had no obviousgrowth abnormalities. The height of the plants was measured at 24 daysafter planting and no significant difference was observed between thewild type plants and the plants containing FIT1 (FIG. 7C). The errorbars indicate the standard deviation of the plant height from theindividual plants (n>8).

Example 4: Introducing FIT1 Homologs into Other Plant Species

The FIT1 sequences isolated and described herein can be introduced intoother plant species to create a plant having resistance or tolerance toa pathogen.

For example, a sequence encoding any one of the proteins of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and functional homologs thereof, orsequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 complementsthereof, fragments thereof, and sequences at least 70% identical theretocan be introduced into a plant to confer resistance or tolerance to apathogen. For example, as described above, Glycine max, which does notpossess a functional FIT1 gene and is susceptible to ASR, may betransformed with a transgene comprising SEQ ID NO: 1, or a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 2, to conferresistance to plant pathogens, such as Phakopsora pachyrhizi, thatsecrete the effector protein AvrFIT1, and cause diseases like ASR. Basedon the transient assays described herein (FIG. 5A-5D), resistance couldalso be achieved with a transgene comprising any one of SEQ ID NOs: 3,5, 7, 9, 11, 13, 15, 17, and/or 19, or a sequence encoding any one ofthe proteins of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and/or 20.

Additional plant species susceptible to ASR or pathogens secretingAvrFIT1-like effector proteins, such as Alysicarpus spp., Astragalusspp., Baptisia spp., Cajanus spp., Calopogonium spp., Caragana spp.,Centrosema spp., Cologania spp., Crotalaria spp., Desmodium spp.,Genista spp., Glycine spp., Glycyrrhiza spp., Indigofera spp.,Kummerowia spp., Lablab spp., Lathyrus spp., Lespedeza spp., Lotus spp.,Lupinus spp., Macroptilium spp., Macrotyloma spp., Medicago spp.,Neonotonia spp., Pachyrhizus spp., Pisum spp., Phaseolus spp.,Pseudovigna spp., Psoralea spp., Robinia spp., Senna spp., Sesbaniaspp., Strophostyles spp., Tephrosia spp., Teramnus spp., Trifolium spp.,Vicia spp., Vigna spp., or Voandzeia spp. could also be transformed withany of the FIT1 sequences disclosed herein to confer resistance to apathogen.

Example 5: Methods of Identifying Pathogen Resistant Genes

FIT1 orthologs are likely present in additional species and generawithin the Fabaceae family. FIT1 orthologs may identified by any numberof means known in the art. This includes sequencing the genome ortranscriptome of a plant species, identifying FIT1 homologs using aBLAST search, identifying putative FIT1 orthologs by constructing aphylogenetic tree of the homologous proteins, and then testing theidentified putative FIT1 genes for AvrFIT1 recognition activity using atransient assay such as that shown in FIG. 5A-5D. Alternatively,synthetic alleles of FIT1 may be designed by combining fragments ofnaturally occurring FIT1 alleles or by introducing amino acidsubstitutions at positions shown to be variable in an alignment offunctional FIT1 proteins and similarly tested for functionality bytransient assay. Methods of alignment of sequences for comparison arewell known in the art. Various programs and alignment algorithms aredescribed in: Smith and Waterman (Adv. Appl. Math., 2:482, 1981);Needleman and Wunsch (J. Mol. Biol., 48:443, 1970); Pearson and Lipman(Proc. Natl. Acad. Sci., 85:2444, 1988); Higgins and Sharp (Gene,73:237-44, 1988); Higgins and Sharp (CABIOS, 5:151-53, 1989); Corpet etal. (Nuc. Acids Res., 16:10881-90, 1988); Huang et al. (Comp. ApplsBiosci., 8:155-65, 1992); and Pearson et al. (Meth. Mol. Biol.,24:307-31, 1994). Altschul et al. (Nature Genet., 6:119-29, 1994)presents a detailed consideration of sequence alignment methods andhomology calculations.

While previous reports have listed some FIT1 homologs as predicted TMVresistance proteins, this is unlikely. FIG. 8 depicts an alignmentbetween the FIT1 alleles from Vigna unguiculata (VuFIT1), Phaseoluslunatus (PlFIT1), Abrus precatorius (ApFIT1), and the N gene, whichgives TMV resistance. The LRR domain is poorly conserved between theFIT1 alleles and the N gene. Therefore, FIT1 is not expected to have thesame activity as the N gene (which recognizes the P50 protein andconfers resistance to Tobacco Mosaic Virus). This prediction can beconfirmed by transient expression of the proteins, which demonstratesthat the N gene can recognize P50 but not AvrFIT1a or AvrFITb and thatFIT1 is not able to recognize P50.

The ability of other potential FIT1 homologs or synthetic genes tofunction for AvrFIT1 perception can be easily and quickly tested usingthe transient expression assays shown and described herein or similarmethods well known in the art. For example, once a FIT1 ortholog isidentified in a plant species that is resistant to ASR, the gene can becloned and tested for effector protein recognition using transientexpression assays and the AvrFIT1a (SEQ ID NO: 23) and AvrFIT1bsequences (SEQ ID NO: 25) described herein (see for example FIGS. 5A-D).Examples of leaf tissue suitable for use in a FIT1-AvrFIT1 assayinclude, but are not limited to, species or accessions of Nicotiana,Solanum, Physalis, Capsicum, Lactuca, Alysicarpus, Astragalus, Baptisia,Cajanus, Calopogonium, Caragana, Centrosema, Cologania, Crotalaria,Desmodium, Genista, Glycine, Glycyrrhiza, Indigofera, Kummerowia,Lablab, Lathyrus, Lespedeza, Lotus, Lupinus, Macroptihum, Macrotyloma,Medicago, Neonotonia, Pachyrhizus, Pisum, Phaseolus, Pseudovigna,Psoralea, Robinia, Senna, Sesbania, Strophostyles, Tephrosia, Teramnus,Trifohum, Vicia, Vigna, and Voandzeia that lack a functional native FIT1gene. Similarly, resistance genes for other pathogens may be identifiedusing this same method, wherein a potential gene is identified, cloned,and tested in transient assays with pathogen effector proteins.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO: 1 shows the nucleic acid sequence of Vigna unguiculata FIT1.

SEQ ID NO: 2 shows the corresponding amino acid sequence of SEQ ID NO:1.

SEQ ID NO: 3 shows the nucleic acid sequence of Vigna radiata FIT1.

SEQ ID NO: 4 shows the corresponding amino acid sequence of SEQ ID NO:3.

SEQ ID NO: 5 shows the nucleic acid sequence of Phaseolus lunatus FIT1.

SEQ ID NO: 6 shows the corresponding amino acid sequence of SEQ ID NO:5.

SEQ ID NO: 7 shows the nucleic acid sequence of Phaseolus vulgaris FIT1.

SEQ ID NO: 8 shows the corresponding amino acid sequence of SEQ ID NO:7.

SEQ ID NO: 9 shows the nucleic acid sequence of Phaseolus acutifoliusFIT1.

SEQ ID NO: 10 shows the corresponding amino acid sequence of SEQ ID NO:9.

SEQ ID NO: 11 shows the nucleic acid sequence of Vigna angularis FIT1.

SEQ ID NO: 12 shows the corresponding amino acid sequence of SEQ ID NO:11.

SEQ ID NO: 13 shows the nucleic acid sequence of Lablab purpureus FIT1.

SEQ ID NO: 14 shows the corresponding amino acid sequence of SEQ ID NO:13.

SEQ ID NO: 15 shows the nucleic acid sequence of Mucuna pruriens FIT1.

SEQ ID NO: 16 shows the corresponding amino acid sequence of SEQ ID NO:15.

SEQ ID NO: 17 shows the nucleic acid sequence of Cajanus cajun FIT1.

SEQ ID NO: 18 shows the corresponding amino acid sequence of SEQ ID NO:17.

SEQ ID NO: 19 shows the nucleic acid sequence of Abrus precatorius FIT1.

SEQ ID NO: 20 shows the corresponding amino acid sequence of SEQ ID NO:19.

SEQ ID NO: 21 shows the nucleic acid sequence of Vigna unguiculataFIT1b.

SEQ ID NO: 22 shows the corresponding amino acid sequence of SEQ ID NO:21.

SEQ ID NO: 23 shows the nucleic acid sequence of Phakopsora pachyrhiziALL40704.1 (AvrFIT1a).

SEQ ID NO: 24 shows the corresponding amino acid sequence of SEQ ID NO:23.

SEQ ID NO: 25 shows the nucleic acid sequence of Phakopsora pachyrhiziALL41167.1 (AvrFIT1b).

SEQ ID NO: 26 shows the corresponding amino acid sequence of SEQ ID NO:25.

SEQ ID NO: 27 shows the nucleic acid sequence of the transientexpression vector with Vigna unguiculata FIT1.

SEQ ID NO: 28 shows the nucleic acid sequence of the transientexpression vector with Phaseolus lunatus FIT1.

SEQ ID NO: 29 shows the nucleic acid sequence of the transientexpression vector with Vigna radiata FIT1.

SEQ ID NO: 30 shows the nucleic acid sequence of the transientexpression vector with Vigna unguiculata FIT1b.

SEQ ID NO: 31 shows the nucleic acid sequence of the transientexpression vector with Vigna angularis FIT1.

SEQ ID NO: 32 shows the nucleic acid sequence of the transientexpression vector with Phaseolus vulgaris FIT1.

SEQ ID NO: 33 shows the nucleic acid sequence of the transientexpression vector with Lablab purpureus FIT1.

SEQ ID NO: 34 shows the nucleic acid sequence of the transientexpression vector with Abrus precatorius FIT1.

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as, an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world.

NUMBERED EMBODIMENTS

-   1. An isolated, recombinant, or synthetic polynucleotide comprising    a nucleic acid sequence encoding a functional FIT1 protein    homologous to SEQ ID NO: 2.-   2. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 70% identity to SEQ ID NO: 2.-   3. The isolated, recombinant, or synthetic polynucleotide of    embodiment 2, wherein the polynucleotide encodes a FIT1 protein from    Abrus precatorius.-   4. The isolated, recombinant, or synthetic polynucleotide of    embodiment 3, wherein the polynucleotide comprises SEQ ID NO: 19, a    polynucleotide encoding SEQ ID NO: 20, complements thereof, or    fragments thereof-   5. The isolated, recombinant, or synthetic polynucleotide of    embodiment 2, wherein the polynucleotide encodes a FIT1 protein from    Cajanus cajan.-   6. The isolated, recombinant, or synthetic polynucleotide of    embodiment 5, wherein the polynucleotide comprises SEQ ID NO: 17, or    a polynucleotide encoding SEQ ID NO: 18.-   7 The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 75% identity to SEQ ID NO: 2.-   8. The isolated, recombinant, or synthetic polynucleotide of    embodiment 7, wherein the polynucleotide encodes a FIT1 protein from    Mucuna pruriens.-   9. The isolated, recombinant, or synthetic polynucleotide of    embodiment 8, wherein the polynucleotide comprises SEQ ID NO: 15, a    polynucleotide encoding SEQ ID NO: 16, complements thereof, or    fragments thereof-   10. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 80% identity to SEQ ID NO: 2.-   11. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 85% identity to SEQ ID NO: 2.-   12. The isolated, recombinant, or synthetic polynucleotide of    embodiment 11, wherein the polynucleotide encodes a FIT1 protein    from Lablab purpureus.-   13. The isolated, recombinant, or synthetic polynucleotide of    embodiment 12, wherein the polynucleotide comprises SEQ ID NO: 13, a    polynucleotide encoding SEQ ID NO: 14, complements thereof, or    fragments thereof-   14. The isolated, recombinant, or synthetic polynucleotide of    embodiment 11, wherein the polynucleotide encodes a FIT1 protein    from Phaseolus lunatus.-   15. The isolated, recombinant, or synthetic polynucleotide of    embodiment 14, wherein the polynucleotide comprises SEQ ID NO: 5, a    polynucleotide encoding SEQ ID NO: 6, complements thereof, or    fragments thereof-   16. The isolated, recombinant, or synthetic polynucleotide of    embodiment 11, wherein the polynucleotide encodes a FIT1 protein    from Phaseolus vulgaris.-   17. The isolated, recombinant, or synthetic polynucleotide of    embodiment 16, wherein the polynucleotide comprises SEQ ID NO: 7, a    polynucleotide encoding SEQ ID NO: 8, complements thereof, or    fragments thereof-   18. The isolated, recombinant, or synthetic polynucleotide of    embodiment 11, wherein the polynucleotide encodes a FIT1 protein    from Phaseolus acutifolius.-   19. The isolated, recombinant, or synthetic polynucleotide of    embodiment 18, wherein the polynucleotide comprises SEQ ID NO: 9, a    polynucleotide encoding SEQ ID NO: 10, complements thereof, or    fragments thereof-   20. The isolated, recombinant, or synthetic polynucleotide of    embodiment 11, wherein the polynucleotide encodes a FIT1 protein    from Vigna radiata.-   21. The isolated, recombinant, or synthetic polynucleotide of    embodiment 20, wherein the polynucleotide comprises SEQ ID NO: 3, a    polynucleotide encoding SEQ ID NO: 4, complements thereof, or    fragments thereof-   22. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 90% identity to SEQ ID NO: 2.-   23. The isolated, recombinant, or synthetic polynucleotide of    embodiment 22, wherein the polynucleotide encodes a FIT1 protein    from Vigna angularis.-   24. The isolated, recombinant, or synthetic polynucleotide of    embodiment 23, wherein the polynucleotide comprises SEQ ID NO: 11, a    polynucleotide encoding SEQ ID NO: 12, complements thereof, or    fragments thereof-   25. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 95% identity to SEQ ID NO: 2.

26. The isolated, recombinant, or synthetic polynucleotide of embodiment1, wherein the polynucleotide encodes a protein having at least 96%identity to SEQ ID NO: 2.

-   27. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 97% identity to SEQ ID NO: 2.-   28. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 98% identity to SEQ ID NO: 2.-   29. The isolated, recombinant, or synthetic polynucleotide of    embodiment 1, wherein the polynucleotide encodes a protein having at    least 99% identity to SEQ ID NO: 2.-   30. An isolated, recombinant, or synthetic polynucleotide comprising    a nucleic acid sequence encoding a FIT1 protein, wherein the protein    is selected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8,    10, 12, 14, 16, 18, 20 and functional homologs thereof.-   31. The isolated, recombinant, or synthetic polynucleotide of    embodiment 30, wherein the polynucleotide comprises a nucleic acid    sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,    7, 9, 11, 13, 15, 17, 19 complements thereof, fragments thereof, and    sequences at least 70% identical thereto.-   32. A genetic construct comprising at least one of the nucleic acid    sequences of any one of embodiments 1-31.-   33. A plant, plant part, or plant cell transformed with at least one    of the nucleic acid sequences of any one of embodiments 1-31 or the    genetic construct of embodiment 32, wherein said plant, plant part    or plant cell is resistant or tolerant to a pathogen.-   34. The plant, plant part, or plant cell of embodiment 33, wherein    the pathogen is a fungus from the order Cantharellales or    Pucciniales.-   35. The plant, plant part, or plant cell of embodiment 33 or 34,    wherein the fungal pathogen is Rhizoctonia solani, Melampsora spp.,    Phakopsora pachyrhizi, Phakopsora meibomiae, Phakopsora euvitis,    Phakopsora spp., Puccinia spp., Uromyces spp., Austropuccinia spp.,    Cronartium spp. or Hemileia vastatrix.-   36. The plant, plant part, or plant cell of any one of embodiments    33-35, wherein the plant, plant part, or plant cell is in the    subfamily Papilionoideae.-   37. The plant, plant part, or plant cell of any one of embodiments    33-36, wherein the plant, plant part, or plant cell is Alysicarpus    spp., Astragalus spp., Baptisia spp., Cajanus spp., Calopogonium    spp., Caragana spp., Centrosema spp., Cologania spp., Crotalaria    spp., Desmodium spp., Genista spp., Glycine spp., Glycyrrhiza spp.,    Indigofera spp., Kummerowia spp., Lablab spp., Lathyrus spp.,    Lespedeza spp., Lotus spp., Lupinus spp., Macroptilium spp.,    Macrotyloma spp., Medicago spp., Neonotonia spp., Pachyrhizus spp.,    Pisum spp., Phaseolus spp., Pseudovigna spp., Psoralea spp., Robinia    spp., Senna spp., Sesbania spp., Strophostyles spp., Tephrosia spp.,    Teramnus spp., Trifolium spp., Vicia spp., Vigna spp., or Voandzeia    spp.-   38. The plant, plant part, or plant cell of any one of embodiments    33-37, wherein the plant, plant part, or plant cell is Glycine max,    and wherein the plant, plant part, or plant cell is resistant to    Asian Soybean Rust caused by Phakopsora pachyrhizi.-   39. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 1, or a nucleotide sequence encoding the amino    acid sequence of SEQ ID NO: 2.-   40. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 3, or a nucleotide sequence encoding the amino    acid sequence of SEQ ID NO: 4.-   41. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 5, or a nucleotide sequence encoding the amino    acid sequence of SEQ ID NO: 6.-   42. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 7, or a nucleotide sequence encoding the amino    acid sequence of SEQ ID NO: 8.-   43. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 9, or a nucleotide sequence encoding the amino    acid sequence of SEQ ID NO: 10.-   44. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 11, or a nucleotide sequence encoding the    amino acid sequence of SEQ ID NO: 12.-   45. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 13, or a nucleotide sequence encoding the    amino acid sequence of SEQ ID NO: 14.-   46. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 15, or a nucleotide sequence encoding the    amino acid sequence of SEQ ID NO: 16.-   47. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 17, or a nucleotide sequence encoding the    amino acid sequence of SEQ ID NO: 18.-   48. The plant, plant part, or plant cell of embodiment 38, wherein    the resistance to Asian Soybean Rust is conferred by a transgene    comprising SEQ ID NO: 19, or a nucleotide sequence encoding the    amino acid sequence of SEQ ID NO: 20.-   49. A method of producing a plant, plant part, or plant cell having    resistance or tolerance to a pathogen, wherein the method comprises:    -   transforming a plant, plant part, or plant cell with a        nucleotide sequence encoding a Toll-like Interleukin-1 Receptor        (TIR) Nucleotide binding, Leucine-rich Repeat (NLR) immune        receptor protein, wherein said immune receptor protein mediates        the perception of pathogen effector protein AvrFIT1 or homologs        thereof; and wherein expression of the immune receptor protein        prevents the pathogen from colonizing the plant, or prevents the        pathogen from affecting plant growth or yield.-   50. The method of embodiment 49, wherein the pathogen effector    protein comprises SEQ ID NO: 24, SEQ ID: 26, or sequences at least    90% identical thereto.-   51. The method of embodiment 49 or 50, wherein the nucleotide    sequence encoding the immune receptor protein has been codon    optimized.-   52. The method of any one of embodiments 49-51, wherein the immune    receptor protein is selected from the group consisting of:    -   an isolated, recombinant, or synthetic polynucleotide comprising        a nucleic acid sequence encoding a FIT1 protein, wherein the        protein is selected from the group consisting of: SEQ ID NOs: 2,        4, 6, 8, 10, 12, 14, 16, 18, 20 and functional homologs thereof,        or    -   an isolated, recombinant, or synthetic polynucleotide encoding a        FIT1 protein, wherein the nucleic acid sequence is selected from        the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,        17, 19 complements thereof, fragments thereof, and sequences at        least 70% identical thereto.-   53. The method of any one of embodiments 49-52, wherein the plant,    plant part, or plant cell is transformed with one or more additional    desired traits.-   54. The method of embodiment 49, wherein the one or more additional    desired traits are stacked together with the immune receptor protein    on the same DNA construct.-   55. The method of any one of embodiments 49-23, further comprising    introgressing one or more additional desired traits.-   56. The method of any one of embodiments 49-55, wherein the one or    more additional desired traits are resistance traits to a disease,    pest, or abiotic stress.-   57. A plant, plant part, or plant cell produced by the method of any    one of embodiments 49-56, wherein the plant, plant part, or plant    cell is resistant to a pathogen.-   58. A plant, plant part, or plant cell produced by the method of any    one of embodiments 49-56, wherein the plant, plant part, or plant    cell is tolerant to a pathogen.-   59. The plant, plant part, or plant cell of embodiment 57 or 58,    wherein the immune receptor protein is transiently expressed.-   60. The plant, plant part, or plant cell of embodiment 57 or 58,    wherein the immune receptor protein is stably expressed.-   61. The plant, plant part, or plant cell of any one of embodiments    49-60, wherein the plant, plant part, or plant cell is in the    subfamily Papilionoideae.-   62. The plant, plant part, or plant cell of embodiment 61, wherein    the plant, plant part, or plant cell is Alysicarpus spp., Astragalus    spp., Baptisia spp., Cajanus spp., Calopogonium spp., Caragana spp.,    Centrosema spp., Cologania spp., Crotalaria spp., Desmodium spp.,    Genista spp., Glycine spp., Glycyrrhiza spp., Indigofera spp.,    Kummerowia spp., Lablab spp., Lathyrus spp., Lespedeza spp., Lotus    spp., Lupinus spp., Macroptilium spp., Macrotyloma spp., Medicago    spp., Neonotonia spp., Pachyrhizus spp., Pisum spp., Phaseolus spp.,    Pseudovigna spp., Psoralea spp., Robinia spp., Senna spp., Sesbania    spp., Strophostyles spp., Tephrosia spp., Teramnus spp., Trifohum    spp., Vicia spp., Vigna spp., or Voandzeia spp.-   63. The plant, plant part, or plant cell of any one of embodiments    57-62, wherein the plant is Glycine max, wherein the plant, plant    part, or plant cell is resistant to Asian Soybean Rust caused by    Phakopsora pachyrhizi.-   64. A method of genetically engineering a pathogen resistance or    tolerance trait in a plant, plant part, or plant cell, comprising:    -   providing a plant species that is susceptible to a pathogen;    -   identifying within the genome of the plant species a homolog of        FIT1, wherein said homolog does not mediate AvrFIT1 recognition;        and    -   genetically modifying a plant, plant part, or plant cell from        the susceptible plant species with targeted gene editing,        wherein said targeted gene editing is directed at the FIT1        homolog, and wherein said targeted gene editing enables the FIT1        homolog to recognize AvrFIT1 and confers resistance or tolerance        to a pathogen.-   65. The method of embodiment 64, wherein the targeted gene editing    uses an engineered or natural nuclease selected from the group    consisting of homing endonucleases/meganucleases (EMNs), zinc finger    nucleases (ZFNs), and transcription activator-like effector    nucleases (TALENs).-   66. The method of embodiment 64 or 65, wherein targeted gene editing    uses a clustered regularly interspaced short palindromic repeats    (CRISPR)-Cas nuclease.-   67. The method of embodiment 66, wherein the nuclease is selected    from the group consisting of Cas9, Cas12, Cas13, CasX, and CasY.-   68. The method of any one of embodiments 64-67, further comprising    breeding with, or asexually propagating the plant.-   69. A genetically modified plant, plant part, or plant cell produced    by the method of any one of embodiments 64-68, wherein said plant,    plant part, or plant cell exhibits resistance or tolerance to a    pathogen.-   70. The genetically modified plant, plant part, or plant cell of    embodiment 69, wherein the pathogen is a fungus from the order    Cantharellales or Pucciniales.-   71. The genetically modified plant, plant part, or plant cell of    embodiment 70, wherein the fungal pathogen is Rhizoctonia solani,    Melampsora spp., Phakopsora pachyrhizi, Phakopsora meibomiae,    Phakopsora euvitis, Phakopsora spp., Puccinia spp., Uromyces spp.,    Austropuccinia spp., Cronartium spp. or Hemileia vastatrix.-   72. The genetically modified plant, plant part, or plant cell of any    one of embodiments 69-71, wherein the fungal pathogen is Phakopsora    pachyrhizi and the plant, plant part, or plant cell is Glycine max.-   73. A method for identifying a functional FIT1 gene and/or allele    thereof comprising:    -   isolating a FIT1 homolog or allele thereof;    -   expressing all or a substantial fragment of said FIT1 homolog or        allele thereof in combination with a homolog of AvrFIT1 in a        plant, plant part, or plant cell; and    -   assaying said plant, plant part, or plant cell for an immune        response.-   74. The method of embodiment 73, wherein the effector protein    comprises SEQ ID NO: 24, SEQ ID: 26, or sequences at least 90%    identical thereto.-   75. The method of embodiment 73 or 74, wherein the FIT1 allele is a    synthetic variant.-   76. The method of any one of embodiments 73-75, wherein the plant,    plant part, or plant cell is a species of Nicotiana, Solanum,    Physalis, Capsicum, Lactuca, Alysicarpus, Astragalus, Baptisia,    Cajanus, Calopogonium, Caragana, Centrosema, Cologania, Crotalaria,    Desmodium, Genista, Glycine, Glycyrrhiza, Indigofera, Kummerowia,    Lablab, Lathyrus, Lespedeza, Lotus, Lupinus, Macroptilium,    Macrotyloma, Medicago, Neonotonia, Pachyrhizus, Pisum, Phaseolus,    Pseudovigna, Psoralea, Robinia, Senna, Sesbania, Strophostyles,    Tephrosia, Teramnus, Trifolium, Vicia, Vigna, and Voandzeia that    lacks a functional native FIT1 gene.-   77. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 1, or a sequence at least 70%    identical thereto.-   78. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 1, or a sequence at least 80%    identical thereto.-   79. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 1, or a sequence at least 90%    identical thereto.-   80. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 2, or an amino acid sequence at least 90%    identical thereto.-   81. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 3, or a sequence at least 70%    identical thereto.-   82. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 3, or a sequence at least 80%    identical thereto.-   83. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 3, or a sequence at least 90%    identical thereto.-   84. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 4, or an amino acid sequence at least 90%    identical thereto.-   85. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 5, or a sequence at least 70%    identical thereto.-   86. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 5, or a sequence at least 80%    identical thereto.-   87. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 5, or a sequence at least 90%    identical thereto.-   88. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 6, or an amino acid sequence at least 90%    identical thereto.-   89. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 7, or a sequence at least 70%    identical thereto.-   90. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 7, or a sequence at least 80%    identical thereto.-   91. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 7, or a sequence at least 90%    identical thereto.-   92. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 8, or an amino acid sequence at least 90%    identical thereto.-   93. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 9, or a sequence at least 70%    identical thereto.-   94. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 9, or a sequence at least 80%    identical thereto.-   95. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 9, or a sequence at least 90%    identical thereto.-   96. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%    identical thereto.-   97. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 11, or a sequence at least 70%    identical thereto.-   98. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 11, or a sequence at least 80%    identical thereto.-   99. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 11, or a sequence at least 90%    identical thereto.-   100. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%    identical thereto.-   101. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 13, or a sequence at least 70%    identical thereto.-   102. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 13, or a sequence at least 80%    identical thereto.-   103. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 13, or a sequence at least 90%    identical thereto.-   104. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%    identical thereto.-   105. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 15, or a sequence at least 70%    identical thereto.-   106. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 15, or a sequence at least 80%    identical thereto.-   107. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 15, or a sequence at least 90%    identical thereto.-   108. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 16, or an amino acid sequence at least 90%    identical thereto.-   109. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 17, or a sequence at least 70%    identical thereto.-   110. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 17, or a sequence at least 80%    identical thereto.-   111. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 17, or a sequence at least 90%    identical thereto.-   112. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%    identical thereto.-   113. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 19, or a sequence at least 70%    identical thereto.-   114. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 19, or a sequence at least 80%    identical thereto.-   115. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising SEQ ID NO: 19, or a sequence at least 90%    identical thereto.-   116. A transgenic plant, plant part, or plant cell having resistance    to Asian Soybean Rust, wherein the resistance is conferred by a    transgene comprising a nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 20, or an amino acid sequence at least 90%    identical thereto.

1.-4. (canceled)
 5. A recombinant DNA construct comprising a nucleicacid sequence, wherein the nucleic acid sequence encodes a proteinselected from the group consisting of SEQ ID NOs: 2, 4, 8, 10, 12, 14,16, 18, 20, and proteins at least 90% identical thereto, and wherein theconstruct is capable of conferring resistance to a fungal pathogen whentransformed into a plant.
 6. A transgenic plant, plant part, or plantcell having resistance or tolerance to a pathogen, wherein theresistance or tolerance is conferred by a transgene comprising a nucleicacid sequence encoding a protein selected from the group consisting ofSEQ ID NOs: 2, 4, 8, 10, 12, 14, 16, 18, 20, and proteins at least 90%identical thereto.
 7. The transgenic plant, plant part, or plant cell ofclaim 6, wherein the pathogen is a fungus from the order Cantharellalesor Pucciniales.
 8. The transgenic plant, plant part, or plant cell ofclaim 7, wherein the fungal pathogen is Rhizoctonia solani, Melampsoraspp., Phakopsora pachyrhizi, Phakopsora meibomiae, Phakopsora euvitis,Phakopsora spp., Puccinia spp., Uromyces spp., Austropuccinia spp.,Cronartium spp., Austropuccinia spp., Cronartium spp. or Hemileiavastatrix.
 9. The transgenic plant, plant part, or plant cell of claim6, wherein the plant, plant part, or plant cell is in the subfamilyPapilionoideae.
 10. The transgenic plant, plant part, or plant cell ofclaim 6, wherein the plant, plant part, or plant cell is Alysicarpusspp., Astragalus spp., Baptisia spp., Cajanus spp., Calopogonium spp.,Caragana spp., Centrosema spp., Cologania spp., Crotalaria spp.,Desmodium spp., Genista spp., Glycine spp., Glycyrrhiza spp., Indigoferaspp., Kummerowia spp., Lablab spp., Lathyrus spp., Lespedeza spp., Lotusspp., Lupinus spp., Macroptilium spp., Macrotyloma spp., Medicago spp.,Neonotonia spp., Pachyrhizus spp., Pisum spp., Phaseolus spp.,Pseudovigna spp., Psoralea spp., Robinia spp., Senna spp., Sesbaniaspp., Strophostyles spp., Tephrosia spp., Teramnus spp., Trifolium spp.,Vicia spp., Vigna spp., or Voandzeia spp.
 11. The transgenic plant,plant part, or plant cell of claim 10, wherein the plant, plant part, orplant cell is Glycine max, and wherein the plant, plant part, or plantcell is resistant to Asian Soybean Rust caused by Phakopsora pachyrhizi.12. The transgenic plant, plant part, or plant cell of claim 11, whereinthe resistance to Asian Soybean Rust is conferred by a transgenecomprising a sequence selected from the group consisting of SEQ ID NOs:1, 3, 7, 9, 11, 13, 15, 17, 19, and sequences at least 70% identicalthereto.
 13. The transgenic plant, plant part, or plant cell of claim11, wherein the resistance to Asian Soybean Rust is conferred by atransgene comprising a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 2, or an amino acid sequence at least 90%identical thereto.
 14. A method of producing a plant, plant part, orplant cell having resistance or tolerance to a pathogen, wherein themethod comprises: transforming a plant, plant part, or plant cell withan isolated, recombinant, or synthetic polynucleotide comprising: anucleic acid sequence encoding all or a substantial fragment of afunctional FIT1 protein, wherein the protein is at least 70% identicalto a protein selected from the group consisting of: SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, and 20, or an isolated, recombinant, or syntheticpolynucleotide encoding a functional FIT1 protein, wherein the nucleicacid sequence is selected from the group consisting of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19 complements thereof, fragments thereof, andsequences at least 70% identical thereto; and selecting a plantcomprising the polynucleotide and having resistance or tolerance to apathogen.
 15. The method of claim 14, wherein the nucleotide sequenceencoding the FIT1 protein has been codon optimized.
 16. The method ofclaim 14, wherein the plant, plant part, or plant cell is transformedwith two or more polynucleotides encoding different FIT1 proteins. 17.The method of claim 14, wherein the plant, plant part, or plant cell istransformed or introgressed with one or more additional desired traits.18. The method of claim 17, wherein the one or more additional desiredtraits are resistance traits to a disease, pest, or abiotic stress. 19.A plant, plant part, or plant cell produced by the method of claim 14,wherein the plant, plant part, or plant cell is resistant or tolerant toa pathogen.
 20. The plant, plant part, or plant cell of claim 19,wherein the FIT1 protein is transiently expressed.
 21. The plant, plantpart, or plant cell of claim 19, wherein the FIT1 protein is stablyexpressed.
 22. The plant, plant part, or plant cell of claim 19, whereinthe plant, plant part, or plant cell is in the subfamily Papilionoideae.23. The plant, plant part, or plant cell of claim 19, wherein the plant,plant part, or plant cell is Alysicarpus spp., Astragalus spp., Baptisiaspp., Cajanus spp., Calopogonium spp., Caragana spp., Centrosema spp.,Cologania spp., Crotalaria spp., Desmodium spp., Genista spp., Glycinespp., Glycyrrhiza spp., Indigofera spp., Kummerowia spp., Lablab spp.,Lathyrus spp., Lespedeza spp., Lotus spp., Lupinus spp., Macroptihumspp., Macrotyloma spp., Medicago spp., Neonotonia spp., Pachyrhizusspp., Pisum spp., Phaseolus spp., Pseudovigna spp., Psoralea spp.,Robinia spp., Senna spp., Sesbania spp., Strophostyles spp., Tephrosiaspp., Teramnus spp., Trifolium spp., Vicia spp., Vigna spp., orVoandzeia spp.
 24. The plant, plant part, or plant cell of claim 19,wherein the plant is Glycine max, wherein the plant, plant part, orplant cell is resistant to Asian Soybean Rust caused by Phakopsorapachyrhizi.
 25. The plant, plant part, or plant cell of claim 19,wherein the resistance to Asian Soybean Rust is conferred by a transgenecomprising SEQ ID NO: 1, or a sequence at least 70% identical thereto.26. The plant, plant part, or plant cell of claim 19, wherein theresistance to Asian Soybean Rust is conferred by a transgene comprisinga nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2,or an amino acid sequence at least 90% identical thereto.
 27. A methodof genetically engineering a pathogen resistance or tolerance trait in aplant, plant part, or plant cell, comprising: providing a plant speciesthat is susceptible to a pathogen; identifying within the genome of theplant species a homolog of FIT1, wherein said homolog does not mediateAvrFIT1 recognition; and genetically modifying a plant, plant part, orplant cell from the susceptible plant species with targeted geneediting, wherein said targeted gene editing is directed at the FIT1homolog, and wherein said targeted gene editing enables the FIT1 homologto recognize AvrFIT1 and confers resistance or tolerance to a pathogen.28. The method of claim 27, wherein the targeted gene editing uses anengineered or natural nuclease selected from the group consisting ofhoming endonucleases/meganucleases (EMNs), zinc finger nucleases (ZFNs),and transcription activator-like effector nucleases (TALENs).
 29. Themethod of claim 27, wherein targeted gene editing uses a clusteredregularly interspaced short palindromic repeats (CRISPR)-Cas nucleaseselected from the group consisting of Cas9, Cas12, Cas13, CasX, andCasY.
 30. A genetically modified plant, plant part, or plant cellproduced by the method claim 27, wherein said plant, plant part, orplant cell exhibits resistance or tolerance to a pathogen.